Gas chromatography-mass spectrometry-based plasma metabolomics analysis in hypertensive patients with Yin deficiency and Yang hyperactivity syndrome

Abstract

BACKGROUND

Hypertension is a significant global health concern and serves as a critical risk factor for cardiovascular diseases, stroke, and renal failure. Traditional Chinese Medicine (TCM) has been utilized for an extended period to address hypertension, with the syndrome known as Yin deficiency and Yang hyperactivity syndrome (YDYHS) frequently observed in individuals with elevated blood pressure. This syndrome is characterized by symptoms including dizziness, tinnitus, irritability, and insomnia, which are thought to result from an imbalance between Yin, representing the cooling and nourishing aspects, and Yang, denoting the active and warming aspects of the body. Despite the prevalent application of TCM in clinical settings, the metabolic mechanisms underlying the YDYHS in the context of hypertension remain inadequately elucidated.

AIM

To explore the differential plasma metabolites and associated pathways in hypertensive patients with YDYHS using gas chromatography-mass spectrometry (GC-MS) to elucidate the distinctive blood metabolite pattern in this patient population.

METHODS

GC-MS was used to analyze plasma samples from 51 hypertensive patients with YDYHS and 20 healthy controls. Chemometric methods, including principal component analysis and partial least squares discriminant analysis, were employed to identify potential biochemical patterns. Simultaneously, the high-quality Kyoto Encyclopedia of Genes and Genomes metabolic pathways database was used to identify associated metabolic pathways. Using variable importance in projection and receiver operating characteristic curve analyses, potential biomarkers were extracted to assess their clinical utility.

RESULTS

Metabolomic profiling of hypertensive patients with YDYHS identified 20 potential biomarkers (4-hydroxybenzoic acid, pectin, 1,2,3-trihydroxybenzene, D-ribose, 3-hydroxybutyric acid, quinic acid, L-lysine, oleic acid, 2-hydroxybutyric acid, linoleic acid, citric acid, alpha-tocopherol, D-glucuronic acid, glycerol, N-acetyl-L-aspartic acid, beta-mannosylglycerate, indolelactic acid, L-glutamic acid, D-maltose, L-aspartic acid) and four metabolic pathways (linoleic acid metabolism; alanine, aspartate and glutamate metabolism; D-glutamine and D-glutamate metabolism; arginine biosynthesis). The identified differential metabolites may serve as potential biomarkers for distinguishing hypertensive patients with YDYHS from healthy controls. The area under the curve values ranged from 0.750 to 0.866. Receiver operating characteristic curve analysis showed that these differential metabolites can effectively classify hypertensive patients with YDYHS and healthy individuals.

CONCLUSION

The metabolomic analysis revealed a distinct blood metabolite pattern in hypertensive patients with YDYHS compared to the healthy control group, highlighting the potential role of the identified 20 biomarkers and four metabolic pathways in these patients. These findings may serve as an important material basis for understanding the occurrence and development of the disease, providing a scientific foundation for future clinical diagnosis.

Key Words: Hypertension; Metabolomics; Gas chromatography-mass spectrometry; Yin deficiency and Yang hyperactivity syndrome; Syndrome differentiation

Core Tip: Metabolomic profiling of hypertensive patients with Yin deficiency and Yang hyperactivity syndrome identified 20 potential biomarkers and four metabolic pathways. These biomarkers and pathways distinguished hypertensive patients with Yin deficiency and Yang hyperactivity syndrome from healthy controls. These potential biomarkers and pathways suggest the underlying metabolic basis of Yin deficiency and Yang hyperactivity syndrome in hypertensive patients. The metabolomic profiles provide novel insights into the mechanisms of Yin deficiency and Yang hyperactivity syndrome in hypertensive patients.

INTRODUCTION

Hypertension is a global public health issue, posing a significant burden on society. Long-term and persistent hypertension, which can result in target organ damage or failure, accounts for approximately 10.4 million deaths worldwide annually and contributes to around 10% of the total healthcare expenditure globally[1]. In comparison to solely relying on Western medicine, the integration of Chinese and Western medicine offers a safe and effective approach to reducing blood pressure in Chinese patients with essential hypertension, improving clinical symptoms without increasing the incidence of adverse events[2].

In Traditional Chinese Medicine (TCM), hypertension is classified under the categories of “vertigo” and “headache”. The target organ damage caused by hypertension has garnered increasing attention in clinical practice, aligning with the TCM pathogenesis theory that emphasizes liver and kidney disorders. The development of hypertension is linked to an imbalance between vasodilation (Yin) and vasoconstriction (Yang). Through a review of 2910 studies on TCM treatment for hypertension, 47 common syndromes have been identified; among them, Yin deficiency and Yang hyperactivity syndrome (YDYHS) (25.25%) is the most prevalent, followed by liver-Yang hyperactivity syndrome (15.7%) and phlegm-dampness congestion syndrome (PDCS) (8.59%)[3]. As a major pathogenetic factor in hypertension, YDYHS has also been included in Chinese guidelines for hypertension management. However, syndrome differentiation often depends on the subjective experience of the physician and lacks standardized rules.

In 2020, several key technology seminars on the development of new drugs in TCM were organized in China, focusing on how to achieve objectivity in TCM evidence. Chinese medicine summarizes the “syndrome” of diseases through observation, olfaction, inquiry, and palpation, indicating the complexity and integrity of the “syndrome”[4]. Metabolomics enables the acquisition of individualized disease management insights by measuring biological samples like blood, urine, and saliva to obtain each patient's metabolic profile[5], which, to some extent, aligns with the TCM principle of "treatment based on syndrome differentiation." Previous studies have shown that 33 potential biomarkers, such as androstenedione and lyso-phosphatidylcholine, and 16 related metabolic pathways, such as steroid hormone and lipid metabolism, are present in prehypertensive patients with excessive liver fire syndrome[6]. Compared with the control group, the contents of acetone, very-low-density lipoprotein, and low-density lipoprotein in hypertension patients with PDCS increased, while the contents of lactate, serine, glucose, methionine, and alanine decreased. Abnormalities were found in lipid metabolism, amino acid metabolism, and glucose metabolism[7]. Although there were studies on YDYHS[8,9] the sample sizes were less than 20 cases.

Herein, we aimed to further expand the sample size to investigate changes in metabolites and metabolic pathways in the plasma of hypertensive patients with YDYHS. Additionally, this study sought to explore the mechanisms of YDYHS in patients with high blood pressure. Plasma metabolomics methods based on gas chromatography-mass spectrometry (GC-MS) and multiple bioinformatics tools were applied to analyze plasma samples from hypertensive patients with YDYHS and healthy controls (HCs). The results obtained will contribute to revealing the metabolic basis of YDYHS in hypertensive patients (Figure 1).

Figure 1 Graphical abstract. ALT: Alanine transaminase; AST: Aspartate transaminase; GGT: Gamma-glutamyl transferase; BUN: Blood urea nitrogen; Glu: Glucose; WBC: White blood cells; LC-MS: Liquid chromatography-mass spectrometry.

MATERIALS AND METHODS

Participants and selection criteria

All participants provided written informed consent. All samples were collected from the Institute of Hypertension or Department of Cardiology, Affiliated Hospital of Nanjing University of Chinese Medicine. Patients who met the inclusion criteria were enrolled in the study. Diagnosis was provided by the same physician. This study was approved by the Ethics Committee at the Affiliated Hospital of Nanjing University of Chinese Medicine (2019NL-190-02).

The inclusion criteria for the experimental group were as follows: (1) Met the diagnostic criteria for primary hypertension according to the Chinese Guidelines for the Prevention and Treatment of Hypertension 2018 Revision[10], and hypertensive patients with blood pressure < 140/90 mmHg after treatment with antihypertensive drugs; (2) Met the YDYHS diagnostic criteria from the Clinical Research Guidelines for New Drugs in Chinese Medicine (2002). Primary diagnostic criteria for YDYHS were the presence of at least two primary (vertigo; headache; soreness of the waist; weakness of the knees; and dysphoria with feverish sensation in chest, palms, and soles) and two secondary manifestations (palpitation; tinnitus; insomnia; and forgetfulness); (3) Aged 35–70 years; and (4) Voluntarily signed an informed consent form.

The inclusion criteria for the control group were as follows: Good previous physical fitness, no history of hypertension, coronary heart disease, diabetes, etc., and history taking, physical examination, and laboratory tests at the time of consultation were within the normal range or showed abnormalities without clinical significance. Cases were selected from the physical examination center or Phase I ward, Affiliated Hospital of Nanjing University of Chinese Medicine.

The exclusion criteria for the experimental group were as follows: (1) Secondary hypertension; (2) Serious life-threatening diseases such as acute myocardial infarction, stroke, heart failure (NYHA Class IV), and malignant arrhythmia; (3) Liver function indexes [aspartate aminotransferase (AST) or alanine aminotransferase (ALT)] greater than two times the upper limit of normal; (4) Patients with a history of mental illness or legal disability; and (5) Participation in other clinical trials.

GC-MS analysis

Instrument: Trace 1310-TSQ8000 Evo Triple quadrupole gas chromatography-mass spectrometry (Thermo Fisher Scientific, United States); Allegra 64R high-speed refrigerated centrifuge (Beckman Coulter, United States); Vortex-Genie 2 vortex mixer (Scientific Industries, United States); BSA124S-CW analytical balance (Sartorius, Germany); Savant SPD1010 vacuum centrifuge concentrator (Thermo Fisher Scientific, United States).

Reagents: Methoxylamine hydrochloride (Sigma-Aldrich, Batch No. WXBD0638V); pyridine (Sigma-Aldrich, Batch No. STBJ2924); 1,2-¹³C myristic acid (Sigma-Aldrich, Batch No. MBC6775); N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) (Sigma-Aldrich, Batch No. BCCD2584); methanol (Merck, Batch No. I1150307122).

Sample processing

Samples were thawed on ice. A 100 μL aliquot was transferred to a 1.5 mL centrifuge tube, and 400 μL of methanol solution (containing 12.5 μg/mL of 1,2-¹³C myristic acid) was added, vortexed for 3 min, and then centrifuged at 4 °C for 10 min at 13000 rpm. Then, 100 μL of supernatant was evaporated in a centrifuge concentrator (45 °C, 15 kPa). To the dried residue, 30 μL of methoxylamine hydrochloride in pyridine solution (10 mg/mL) was added, vortexed for 5 min, and incubated at 30 °C for 1.5 h. Subsequently, 30 μL of BSTFA (containing 1% TMCS) was added, vortexed for 1 min, and incubated at 37 °C for 0.5 h. After derivatization, the mixture was centrifuged at 13000 rpm for 10 min, and 40 μL of the supernatant was transferred to a sample vial for GC-MS analysis.

Quality control processing

Aliquots from all study samples were pooled. A 100 μL volume of this pooled mixture was used as a quality control (QC) sample and processed according to the steps described above for individual samples.

Analytical conditions

A TG-5MS capillary column (0.25 mm × 30 m × 0.25 μm) was used. The temperature program was: 0–1 min, 60 °C; 1–14 min, ramp from 60 °C to 320 °C (specify rate if available, e.g., at 20 °C/min); 14–19 min, 320 °C. The split ratio was 20:1, and the injection volume was 1 μL. Mass spectrometry was performed in full scan mode with a scan range of m/z 50–500. The carrier gas was high-purity helium (purity > 99.999%) at a flow rate of 1.2 mL/min.

Data processing

The spectral data obtained by GC-MS were processed using MS-DIAL software, and metabolites were identified against the National Institute of Standards and Technology (NIST) database. The data matrices were analyzed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) using the MetaboAnalyst website (https://www.metaboanalyst.ca) to identify differential metabolites and associated metabolic pathways. Receiver operating characteristic curves of the differential metabolites were plotted using MedCalc software to assess their discriminatory power.

Statistical analysis

For quantitative data, those following a normal distribution are expressed as the mean ± SD, and an independent samples t-test was used for comparisons. Data not following a normal distribution are expressed as median and interquartile range [median (IQR: P25–P75)], and the Wilcoxon rank sum test was used for comparisons. Qualitative data, presented as counts (n) and percentages (%), were compared between groups using the χ² test (or Fisher's exact test, as appropriate). Data were processed and analyzed using SPSS 25.0 software, and P < 0.05 was considered statistically significant.

RESULTS

Baseline comparison

A total of 71 participants were recruited and divided into the hypertension (YDYHS) group (n = 51) and the HC group (n = 20). The baseline comparisons are summarized in Table 1. The two groups showed statistically significant differences in white blood cell count (WBC), ALT, AST, gamma-glutamyl transferase (GGT), blood urea nitrogen (BUN), and blood glucose (Glu) (P < 0.05).

Table 1 Baseline comparison.

Project	Healthy subjects	Hypertension (YDYHS)	Normal reference	t/Z	P value
WBC (× 109/L)	5.4860 ± 1.04219	6.7019 ± 1.52323	4.00-10.00	3.254	0.002a
RBC (× 1012/L)	4.7685 ± 0.41509	4.8728 ± 0.50817	4.00-5.50	0.809	0.422
Hb (g/L)	141.85 ± 14.147	145.79 ± 13.779	120-160	1.062	0.292
PLT (× 109/L)	236.45 ± 51.953	235.53 ± 47.571	100-300	-0.070	0.944
ALT (U/L)	14 (12, 17)	22 (15, 27)	< 42	-3.677	0.000a
AST (U/L)	17 (15, 19)	18 (16, 24)	< 38	-2.315	0.021a
TBil (μmol/L)	11.41 (8.84, 16.28)	11.10 (9.19, 15.28)	5.10-28.00	-0.319	0.750
ALP (U/L)	77.45 ± 18.349	83.12 ± 22.374	45-125	1.005	0.319
GGT (U/L)	14 (12, 19)	22 (16, 30)	11-50	-3.232	0.001a
CR (μmol/L)	72.4 (59.9, 84.4)	67.1 (59.8, 76.6)	44.0-110.0	-0.917	0.359
BUN (mmol/L)	4.12 (3.51, 4.87)	4.96 (4.13, 5.90)	2.86-8.20	-2.899	0.004a
Glu (mmol/L)	4.54 (4.31, 4.86)	5.54 (5.21, 5.95)	3.89-6.11	-5.474	0.000a

^aP < 0.05, there is statistical difference between the two groups. ALT: Alanine transaminase; AST: Aspartate transaminase; GGT: Gamma-glutamyl transferase; BUN: Blood urea nitrogen; Glu: Glucose; WBC: White blood cells; RBC: Red blood cells; Hb: Hemoglobin; PLT: Platelets; CR: Creatinine.

Biomarker identification and pathway analysis

QC samples were closely distributed and concentrated (Supplementary Figure 1), and the analytical system was stable during the process. QC total ion flow diagram is shown in Supplementary Figure 2. With the help of Metaboanalyst website to plot PCA and PLS-DA, there was a clear separation between the two groups of metabolites (Figure 2), suggesting the existence of metabolic differences between the two groups. The volcano plot and heat map of the different metabolites were plotted by setting “P < 0.05, fold change = 1.2” (Figure 3), and the peak ratio of metabolites in the disease group/healthy group is expressed by fold change.

Figure 2 Serum metabolic patterns changed in hypertensive patients compared to healthy subjects. A: Partial least squares discriminant analysis; B: Model validation.

Figure 3 Volcano map of differential metabolites. FC: Fold change.

After analysis, 20 differential metabolites existed in the two groups, including 14 up-regulated and 6 down-regulated metabolites (Table 2), mainly involving five metabolic pathways, including lipid metabolism and amino acid metabolism (Table 3 and Figure 4). The ROC curves of differential metabolites were further plotted using MedCalc software (Supplementary Figure 3), and the AUC values were distributed in the range of 0.684-0.866.

Figure 4 Metabolic pathways of 20 differential metabolites.

Table 2 Differential metabolites between the two groups.

No.	Var ID	HMDB	KEGG	FC	P value	PubChem	Trend
1	4-Hydroxybenzoic acid	HMDB0000500	C00156	11.566	< 0.01	135	↑
2	Pectin	HMDB0003402	C08348	3.1576	< 0.01	65550	↑
3	1,2,3-Trihydroxybenzene	HMDB0013674	C01108	2.7443	0.033	3446	↑
4	D-Ribose	HMDB0000283	C00121	2.6621	< 0.01	5779	↑
5	3-Hydroxybutyric acid	HMDB0000357	C01089	2.2031	0.011	441	↑
6	Quinic acid	HMDB0003072	C00296	1.8801	0.034	6508	↑
7	L-Lysine	HMDB0000182	C00047	1.6739	< 0.01	6508	↑
8	Oleic acid	HMDB0000207	C00712	1.5228	≤ 0.01	445639	↑
9	2-Hydroxybutyric acid	HMDB0000008	C05984	1.4935	0.01	11266	↑
10	Linoleic acid	HMDB0000673	159	14928	0.040	5280450	↑
11	Citric acid	HMDB0000094	C00158	1.4718	0.01	311	↑
12	Alpha-tocopherol	NA	NA	1.375	0.01	NA	↑
13	D-Glucuronic acid	HMDB0000127	C00191	1.3608	< 0.01	94715	↑
14	Glycerol	HMDB0000131	C00116	1.3121	< 0.01	753	↑
15	N-Acetyl-L-aspartic acid	HMDB0000812	C01042	0.48705	0.01	65065	↓
16	Beta-mannosylglycerate	NA	NA	0.59765	< 0.01	NA	↓
17	Indolelactic acid	HMDB0000671	C02043	0.65739	0.011	92904	↓
18	L-Glutamic acid	HMDB0000148	C00025	0.66721	0.011	33032	↓
19	D-Maltose	HMDB0000163	C00208	0.69193	0.01	10991489	↓
20	L-Aspartic acid	HMDB0000191	C00049	0.70828	0.042	5960	↓

According to substance comparison, numbers 2, 3, 4, 5, 7, 13, 17, 18, 19, and 20 correspond to lyxose, pyrogallol, ribose, betahydroxybutyric acid, lysine, glucuronic acid, indole-3 act, glutamic acid, maltose 1, and aspartate, respectively. FC: Fold change.

Table 3 Metabolic pathways of differential metabolites.

Pathway	Hits/Total	Expected	Impact	P value
Linoleic acid metabolism	1/5	0.041935	1	0.041
Alanine, aspartate, and glutamate metabolism	4/28	0.23484	0.50722	< 0.01
D-Glutamine and D-glutamate metabolism	1/6	0.050323	0.5	0.049
Arginine biosynthesis	2/14	0.11742	0.11675	< 0.01

DISCUSSION

In the baseline comparison, values of all safety indicators were within normal ranges. The YDYHS group showed statistically significant increases in WBC, ALT, AST, GGT, BUN, and Glu when compared with the HC group. In TCM, YDYHS-type hypertension is characterized by insufficient Yin fluid, resulting in a relative Yang excess, causing hypertension symptoms. Changes in WBC in these patients may be associated with their immune status and inflammatory responses. An elevated WBC count may correlate with chronic inflammation, metabolic syndrome, and an increased risk of cardiovascular diseases. This suggests that a certain level of inflammation may contribute to alterations in WBC levels. The increases in ALT, AST, GGT, BUN, and Glu observed in YDYHS-type hypertensive patients indicate that these patients are more likely to experience damage to organ function and target organs compared to healthy individuals. The results suggest that hypertensive patients with YDYHS may have overactivated inflammatory reactions, liver metabolic disorders, potential for hyperhomocysteinemia (inferred from BUN), and blood glucose abnormalities that can distinguish them from healthy participants.

A study involving 2935 healthy subjects divided them into three groups based on WBC distribution: Group 1 (< 4.7 × 10⁹/L), group 2 (4.7-5.999 × 10⁹/L), and group 3 (≥ 6.0 × 10⁹/L). After an average follow-up of 4.5 years, 908 subjects had systolic blood pressure ≥ 140 mmHg. Compared to group 1, the risk ratio of hypertension in group 2 was 1.07 (95% confidence interval: 0.90-1.26), with a higher risk ratio in group 3. It suggests that the incidence rate of hypertension increases with the leukocyte count, which may promote the process of atherosclerosis through leukocyte adherence to endothelial cells, leading to an increase in systemic arterial stiffness and causing hypertension[11]. A study investigating the potential correlation between WBC count and blood pressure levels revealed that an elevated WBC count may serve as an independent risk factor for hypertension, implying a significant role for inflammation in the pathophysiological mechanisms underlying hypertension. Consequently, incorporating WBC count as an indicator in clinical practice can facilitate early intervention and management strategies for assessing the risk of hypertension more effectively[12].

One cross-sectional study evaluated the association between elevated liver enzymes and hypertension. The study found that compared with the normal blood pressure group, the average concentrations of serum ALT, AST, and GGT in the hypertension group were significantly increased, indicating a relatively higher incidence of elevated liver enzymes in patients with hypertension[13]. Another large sample study of 21293 healthy participants, followed up for 3.9 years (average) with hypertension as the observation endpoint, reached similar conclusions. In addition, in the subgroup analysis of participants with ALT ≤ 2 times the normal limit (80 U/L) (n = 20983), this study showed that high ALT and GGT were significantly associated with the incidence of hypertension[14]. Hyperhomocysteinemia and dysglycemia are recognized risk factors for hypertension. BUN is associated with homocysteine levels. A study has shown that BUN serves as an independent risk factor in male hypertensive patients[15]. Another study found that compared to a lower baseline of Glu (< 5.6 mmol/L), women with a higher baseline of Glu (≥ 5.6 mmol/L and < 7.0 mmol/L) had a significantly increased risk of developing new-onset hypertension[16]. Additionally, a 2019 study indicated that fasting blood glucose serves as a predictive factor for future hypertension in middle-aged Japanese men and women, which supports fasting blood glucose as an effective biomarker for identifying the risk of hypertension in middle-aged populations and emphasizes the importance of early management and intervention in preventing cardiovascular diseases. The results of our study align with these previous findings[17].

4-Hydroxybenzoic acid, also known as p-hydroxybenzoic acid, has anti-inflammatory, antioxidant, cardioprotective, and vasodilatory effects. As an important target of Lei-gong-gen formula granule in the intervention of hypertension, 4-hydroxybenzoic acid exhibits good binding ability with NOS3, SRC, PIK3CA, and AKT, which may act on vascular smooth muscle by stimulating endothelial cells to release nitric oxide (NO), further promoting vasodilation and lowering blood pressure[18]. There are various known forms of hydroxybenzoic acid compounds, such as 2,3-dihydroxybenzoic acid (pyrocatechuic acid), 2,5-dihydroxybenzoic acid (gentisic acid), and 3,4-dihydroxyphenylacetic acid (protocatechuic acid), which can activate the Nrf2 signaling pathway, increase the expression of antioxidant enzymes, and reduce hypertensive endothelial dysfunction caused by oxidative stress[19]. This suggests that 4-hydroxybenzoic acid may also protect the vascular endothelium and alleviate hypertension similarly. 4-Hydroxybenzoic acid may be in a relatively balanced state in normal individuals, with a compensatory increase in the disease state for self-regulation.

Compared with HCs, the levels of linoleic acid (LA) were increased in hypertensive patients with YDYHS. LA can increase the risk of cardiovascular disease by inducing arachidonic acid to promote inflammation[20], leading to increased oxidized LA metabolites, inducing the formation of foam cells and causing endothelial cell dysfunction[21]. In addition, LA induces the expression of vascular cell adhesion molecule-1 in endothelial cells through nuclear factor-kappaB signal transduction, activating the proinflammatory response of vascular endothelial cells and leading to atherosclerosis[22]. Vascular endothelial dysfunction leads to the interaction between atherosclerosis and hypertension, which is the central link in the event chain where hypertension further leads to damage to important target organs such as the heart, brain, and kidney. It is speculated that patients with YDYHS may have more prominent vascular endothelial damage problems than healthy individuals, which affects the occurrence and development of hypertension. LA is also the most prevalent fatty acid found in arterial plaques, and its stable oxidation product—hydroxyoctadecadienoic acid—serves as a marker of oxidative stress and a component of oxidized lipids in atherosclerosis (AS). This compound promotes the progression of AS and increases the risk of cardiovascular events[21]. Based on these findings, this study suggests that LA may exert a bidirectional regulatory effect. Furthermore, it is possible that LA has upstream or downstream activators that play a dual role in the cardiovascular system, warranting further investigation.

The alanine, aspartate, and glutamate metabolism pathway is a key pathway in regulating oxidative stress in cardiovascular diseases[23]. Oxidative stress is a pathological process that occurs when there is an imbalance between oxidation and antioxidant activity in the body. Moderate levels of reactive oxygen species (ROS) play an important role in cell signaling, but excessive levels of ROS can lead to damage to proteins, lipids, and even cell death. Oxidative stress is one of the mechanisms of hypertension, which leads to dysfunction of the vascular endothelium, destruction of vascular structure, and acceleration of the atherosclerosis process, and finally causes hypertension. Elevated levels of aspartic acid and its metabolites, including quinolinic acid, in patients with hypertension are linked to a heightened risk of cardiovascular disease. Quinolinic acid, the final product of tryptophan metabolism, is closely associated with inflammation, oxidative stress, and endothelial dysfunction[24]. Abnormal glutamate metabolism impacts vascular function by affecting the proliferation and migration of vascular smooth muscle cells. This mechanism is particularly evident in pulmonary hypertension and other cardiovascular diseases. Future research should further investigate the specific pathways of glutamate metabolism and its role in various cardiovascular conditions to develop more effective therapeutic strategies[25]. The decrease in alanine, aspartic acid, and glutamate metabolites in patients with YDYHS suggests the activation of oxidative stress, which may lead to excessive consumption of these metabolites or inhibition of antioxidant production in pathological conditions.

Glutamine is a precursor substance of glutamate. Glutamine first enters the cell through transporters and then is converted into glutamate in mitochondria through the catalytic action of glutaminase. Glutamate is further converted into α-ketoglutaric acid through glutamate dehydrogenase, alanine transaminase, or aspartate transaminase. α-Ketoglutaric acid is an important intermediate product in the tricarboxylic acid cycle, and metabolic disorders of this substance can affect energy metabolism in the body[26]. A study has shown that direct injection of glutamic acid into the cerebral cistern of experimental dogs resulted in an increase in blood pressure. Further analysis suggested that this might be related to the involvement of glutamic acid in central blood pressure regulation and an increase in sympathetic nervous tension[27]. D-Glutamine plays a significant role in angiogenesis through a nitric oxide synthase-dependent mechanism, which promotes the growth and migration of endothelial cells. The metabolism of glutamine is essential for the biosynthesis of L-arginine, thereby influencing the production of endothelium-derived relaxing factor by endothelial cells[28]. Hypertensive patients with YDYHS can also exhibit altered glutamate metabolism, possibly manifested as energy utilization disorders and high sympathetic activity.

Nitric oxide synthase exists in the endothelial cells of blood vessels, and under certain conditions, L-arginine is decomposed into NO and L-citrulline[29]. The arginine metabolic pathway plays a crucial role in the pathogenesis and management of hypertension by regulating NO production and the urea cycle. Endothelial dysfunction, oxidative stress, and vascular remodeling, which arise from metabolic abnormalities, are fundamental mechanisms underlying hypertension. Arginine supplementation and the regulation of enzyme activity offer promising new treatment options. Future research should integrate genetic background and metabolic phenotype to create more targeted intervention strategies[30]. NO is an endothelial relaxing factor that soothes vascular smooth muscle, dilates blood vessels, and regulates blood pressure. Research has shown[31] that oral L-citrulline combined with L-arginine can rapidly enhance the bioavailability of NO, which has certain clinical significance in regulating blood pressure. Conversely, when the arginine biosynthesis pathway is affected, NO generation also decreases, leading to a decrease in vasodilation function and a rise in blood pressure. In addition, L-arginine is an important dietary amino acid. Supplementing L-arginine plays a role in reducing inflammation and oxidative stress pathways, lowering blood pressure, decreasing left ventricular damage, and reducing abdominal fat[25]. The imbalance of arginine biosynthesis may also be one of the characteristic pathways in hypertensive patients with YDYHS.

CONCLUSION

In summary, metabolomic profiling of hypertensive patients with YDYHS identified 20 potential biomarkers and four metabolic pathways. These distinguished hypertensive patients with YDYHS from healthy controls. These potential biomarkers and pathways suggest the substantial metabolic basis of YDYHS in hypertensive patients. The metabolomic profiles highlight modernized insights into the mechanisms of YDYHS in these patients.