
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12460-7
10.1016/j.heliyon.2024.e36429
e36429
Research Article
Daily koumiss has positive regulatory effects on blood lipids and immune system: A metabolomics study
Wang Leqi ab1
Sun Yuanfang bc1
Du Lijing bc
Wang Qian ab
Zhan Min ab
Li Shasha happylishasha@163.com
b⁎⁎
Xiao Xue erxiaohappy@163.com
a⁎
a Institute of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, China
b The Second Clinical College of Guangzhou University of Chinese Medicine, Guangzhou, China
c School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
⁎ Corresponding author. erxiaohappy@163.com
⁎⁎ Corresponding author. happylishasha@163.com
1 These authors contributed equally to this work.

17 8 2024
30 8 2024
17 8 2024
10 16 e364294 11 2023
5 8 2024
15 8 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Koumiss, a traditional Mongolian beverage, is believed to possess high nutritional value and potential medical benefits. However, there is a lack of comprehensive research on its potential impact on the human body. Metabolomics, as a sensitive approach in systems biology, offers a new avenue for studying the overall effects of koumiss. In this work, metabolomics was utilized to identify potential biomarkers and pathways associated with koumiss using UPLC-MS detection, pattern recognition analysis, pathway enrichment, network pharmacology. The findings indicated that koumiss exerts a beneficial regulatory influence on lipid metabolism, neurotransmitters, hormones, phospholipids and arachidonic acid metabolism, besides up regulating the content of nutrients. It could reduce the risks of dyslipidemia and inflammatory responses. This study confirmed the benign regulatory effect of koumiss on normal organism from the perspective of endogenous metabolites, and provided objective support for the promotion and application of this ethnic food.

Keywords

Koumiss
Metabolomics
Lipid metabolism
Cardio-cerebrovascular
==== Body
pmc1 Introduction

Koumiss is a traditional fermented dairy product originating from central Asia. It is made from fresh horse milk fermented by lactic acid bacteria and yeasts spontaneously. Described in the Principles of Correct Diet (Yin Shan Zheng Yao) of the Yuan Dynasty (1271 CE–1368), koumiss is characterized by its light and warm nature, with a taste profile encompassing sweet, sour and some astringent notes. It has the functions of invigorating spleen, nourishing stomach, moistening lung, promoting blood circulation and improving sleep. As a traditional fermented dairy product, koumiss is rich in amino acids, unsaturated fatty acids, lactose, vitamins, minerals and other essential components of the human body, which are beneficial to health [1,2]. Additionally, research suggests that koumiss may have therapeutic properties, including enhancing immune function [3] and serving preventive or adjuvant effect on cardiovascular [4], digestive and kidney diseases [5].

Currently, research on koumiss primarily centers on its probiotic bacterium, including the isolation, identification and activity of microbiota in koumiss [[6], [7], [8], [9], [10]]. However, the effect of koumiss as a daily diet for the normal body remains unclear. Metabolomics is a correlation between data-intensive chemical analysis and chemometrics, used for metabolic profiling in intricate systems [[11], [12], [13]]. It could reflect changes in endogenous metabolites that are the most sensitive to disturbance of external impact on the organism, providing a new perspective on assessing the holistic influence of koumiss. In this study, metabolomic strategy was approached to explore the disturbance of this traditional fermented food on normal body metabolism, accordingly to provide basic data for the daily use of koumiss, and to provide a potential mechanism for its further application in dairy therapy.

2 Materials and methods

2.1 Instruments and reagents

Waters UPLC (Waters, USA); AB SCIEX Triple TOF 5600 (AB Sciex, USA); Milli-Q ultrapure water instrument (Millipore, USA); Velocity 14R high-speed refrigerated centrifuge (Dynamic, Australia); Chromatographic-grade acetonitrile and formic acid (Merck, Germany); AB Sciex APCI calibration solution for Triple TOF (AB Sciex, USA). Koumiss, bought from the Ili basin market in Xinjiang, is kept in a refrigerator at temperatures below −20 °C.

2.2 Animal experiments

Twenty male specific pathogen-free Sprague-Dawley (SD) rats (weight, 180 ± 20 g) were provided by the Experimental Animal Centre of Southern Medical University (certificate number, 44002100013523). All tests were conducted following the globally recognized standard protocols for animal usage. The research was carried out in compliance with the national laws of China and local regulations, and the Ethical Committee of Guangdong Provincial Hospital of Chinese Medicine also approved the treatment and management of the rats. The rats were housed under standard environmental conditions (23 ± 2 °C, 55 % ± 5 % humidity and 12 h/12 h light/dark cycle) and were allowed to eat freely.

After 3 days of adaptive feeding, the rats were randomly divided in two groups: the control group (feeding saline) and the SMN group (feeding koumiss). Koumiss or saline were given at the dose of 22.5 mL/kg/d (calculated as 250 mL of Koumiss per person everyday) by gastric administration for 8 weeks. Rats were sacrificed after being anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg of body weight) (GBCBIO Technologies, Guangzhou, China) and about 3 mL blood was obtained from the abdominal aorta in each rat. Blood was centrifuged at 1500 rpm for 10 min at normal temperature. Serum was collected and immediately stored at −80 °C.

2.3 Sample collection and preparation

Acetonitrile (1200 μL) was combined with thawed serum (300 μL) and stirred for a duration of 2 min. After centrifugation at 15,000 rpm for 15 min at 4 °C, the supernatant was filtered by a microporous filter (0.22 μm) and transferred to HPLC vial. Prior to the sample detection, the precision of the instrument, the stability of the sample and the repeatability of the method were investigated firstly. The RSD values of retention time and peak area were calculated to assess the methodology of metabolomics analysis. During sample sequence detection, QC samples and calibration solution were detected at every 5 injection intervals.

2.4 Instrumentation and conditions

The separation condition for chromatography in UPLC-Q/TOF MS was carried out using an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), with the temperature set at 35 °C. The mobile phase was water (0.1 % formic acid) as phase A and acetonitrile as phase B. Gradient elution was used and set as follows: 0–2 min, 2 % B; 4 min, 25 % B; 10 min, 50 % B; 12 min, 65 % B; 22 min, 85 % B; 28–30 min, 98 % B. The volume of the injection was 5 μL, while the velocity of the flow was 0.4 mL per minute. Each wash cycle consisted of 200 μL of strong wash solvent (80 % MEOH-H2O, 8:2, v/v) and 600 μL of weak wash solvent (10 % MEOH-H2O, 1:9, v/v).

The mass spectrum condition in AB SCIEX Triple TOF 5600 was performed on the Duo Spray source (AB Sciex, UK), as ESI probe for sample analysis and APCI probe for calibration solution. MS spectra were acquired both in positive and negative ion mode with high resolution, and information dependent acquisition (IDA) was used to acquire fragment information of the top 6 compounds automatically. The desolvation and auxiliary gas flow were both 50 mL/min, and curtain gas was 35 mL/min. The ion spray voltage was 5500 V in positive ion mode and 4500 V in negative ion mode respectively at temperature 500 °C. For MS scan, the declustering potential voltage was 80 V in positive mode and 100 V in negative mode with accumulation time of 0.1 s. For product ion scan, the collision energy was 35 V with a spread of 15 V. Dynamic background subtractions were used throughout the acquisition process.

2.5 Instrumentation and conditions

The high stability of the tool and technique offered significant confidence in the dependability and precision of the experimental analysis. Therefore, a range of method validation tests, including precision, repeatability, and stability, were conducted to evaluate the UPLC-Q/TOF MS method. The procedures for preparing and determining the samples were carried out as per the previously mentioned section. The accuracy was evaluated by performing six successive injections of the sample solutions for analysis. In terms of repeatability, six example solutions were concurrently extracted. The stability was examined by studying a single sample over a 24-h duration (at intervals of 0, 4, 8, 12, 16, 20, and 24 h) [13].

2.6 Data processing

The original mass spectrum data were conducted by MarkerView software (version 1.2.1, AB Sciex) to align peaks, reduce noise, normalize and correct missing values. The data acquisition range was 1–30 min, and the minimum peak width was 50 ppm. The deviation of retention time and m/z are within 0.05 min and 25 ppm respectively.

The processed data was then exported into SIMCA-P 13.0 and processed by the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). PCA was used first to determine the general interrelation between the groups as unsupervised analysis, and PLS-DA was subsequently performed to maximize the difference in metabolic profiling as supervised analysis. The fitness qualities of the PLS-DA model were evaluated with value of R2 (the percentage of variation being explained by the model) and Q2 (denoting the predictive ability of the model). A typical 7 round cross validation was used to validate the model against over fitting. The variables with VIP value (variable importance in projection) of PLS-DA over 1.5 and p value of t-test between groups less than 0.05 were deemed to be potential metabolite biomarkers.

Qualitative analysis of compounds was performed using Peak View software 1.2 version (AB Sciex, USA), by searching the databases of HMDB (www.hmdb.ca/spectra/ms/search), Pubchem (pubchem.ncbi.nlm.nih.gov), NIST (www.nist.gov/srd/nist1a.html), MassBank (massbank.eu/MassBank/) and KEGG (www.genome.jp/kegg/) according to the information of MS information, isotope peak ratio and MS/MS fragments. Metabolic pathway analysis was performed based on identified metabolites in the system of MetaboAnalyst (www.metaboanalyst.ca/).

2.7 Statistics and data availability

SPSS software (Version 18.0, USA) was used for statistical analyses. Data were presented as the mean ± standard deviation for continuous variables with a normal distribution. Assumptions of normality and homogeneity of variance were first checked, and independent samples t-test was used to analyze the differences between groups. Statistical differences were acknowledged for p values below 0.05, while p values below 0.01 were deemed to have significant differences.

The entirety of the data produced throughout this research is incorporated in the published paper, and the unprocessed data can be obtained from the author responsible, given a reasonable request.

3 Results

3.1 General observations

In this study, the weight, organ index and some biochemical indicators were monitored to observe the effects of koumiss on the body by daily intervention. Compared to control group, the spleen of rats in SMN group was significantly enlarged (p < 0.05), and the blood triglyceride content was significantly reduced (p < 0.05). In weight, other organ indexes and blood indicators, no significant differences were observed. It is worth noting that the weight and blood glucose content of SMN rats showed a downward trend, no statistical difference though. The spleen index and triglyceride were shown in Fig. 1A and B, and the other indicators were listed in supplementary materials.Fig. 1 The spleen index and blood triglyceride on rats. (A) Spleen index of rats; (B) Blood triglyceride (CON vs SMN: **p < 0.05).

Fig. 1

3.2 Metabolic profiling

In this study, metabolic profiling of serum samples was conducted in both positive and negative ionization modes due to the detection of numerous chromatographic peaks in both modes. Serum samples were conducted UPLC-MS under the optimized conditions and representative profile is shown in Fig. 2A and B.Fig. 2 Represented UPLC/MS BPC profiles on serum samples of rats. (A) Positive ionization mode; (B) Negative ionization mode.

Fig. 2

The paired retention time_m/z of these peaks: 1.35_204.1228, 5.42_608.3840, 12.47_415.2123, 14.01_520.3390, 14.89_496.3390, 16.20_510.3544, 17.09_524.3702, 17.72_524.3705, 23.31_703.5737 were selected in positive ion mode, and 1.34_167.0253, 5.38_324.9539, 11.64_604.3388, 13.99_564.3423, 14.44_540.3422, 14.89_540.3416, 17.08_568.3737, 17.71_568.3726, 20.68_303.2384 were selected in negative ion mode to conduct methodological investigation. The variance of their retention time and peak intensity were calculated respectively. It was found RSD values of retention time and peak intensity were were less than 1 % and 10 %, indicating a reliable and reproducible metabolomics analysis method.

3.3 Pattern recognition

In this study, the significance of the intensity was reduced by Pareto scaling to improve equal importance of variables regardless of the magnitude. PCA was conducted first on the normalized UPLC-MS data to give the comprehensive view of the clustering trend (Fig. 3A and B). In PCA score plot, the control and SMN group were separated clearly both in positive and negative ion mode, indicating that koumiss drinking daily could alter endogenous metabolism of rats significantly.Fig. 3 The metabolic path of different rat clusters in the pattern recognition score plot. (A) PCA score plot in positive ionization mode; (B) PCA score plot in negative ionization mode; (C) PLS-DA score plot in positive ionization mode, Q2Y (cum) = 0.949，R2X (cum) = 0.618，R2Y (cum) = 0.999; (D) PLS-DA score plot in negative ionization mode, Q2Y(cum) = 0.929，R2X(cum) = 0.453，R2Y(cum) = 0.994.

Fig. 3

Subsequently, PLS-DA was conducted to maximize the difference in metabolic profiling and to find the metabolites with a significant concentration change as shown in Fig. 3C and D. In the positive mode, the R2Y and Q2Y values calculated by SIMCA-P package were 0.999 and 0.949 respectively, meaning 99.9 % of data fit the model and 94.9 % of data could be predicted by this model. In the negative mode, the R2Y and Q2Y values were 0.994 and 0.929 respectively, meaning 99.4 % of data fit the model and 92.9 % of data could be predicted by this model. Both the Q2Y and R2Y close to 1 indicate an excellent model which is good to fitness and prediction.

3.4 Potential biomarkers and related pathway

Metabolites that significantly contributed to the clustering and discrimination were identified, with VIP >1.5 in PLS-DA analysis and p < 0.05 in independent samples t-test. Finally, 42 potential biomarkers were identified and listed in Table 1. As shown in Fig. 4A, the biomarkers mainly included nutrients (amino acids, vitamins, DHA, hormones and related productions), metabolites involved in arachidonic acid and cholesterol regulation (bile acids, carnitines and ubiquinol) and cardiovascular and cerebrovascular disease (ceramide, neuromedin, derivatives of linoleic acid, as well as some phospholipids). The relative levels of biomarkers were also analyzed, and the heat map was constructed as shown in Fig. 4C. Metabolic pathway analysis indicated that the influence of koumiss daily on normal rats is mainly related to pathways of glycerophospholipid metabolism, bile acids biosynthesis and metabolism, amino acids metabolism and degradation, as well as vitamins and purine metabolism (Fig. 4B). Besides, network pharmacology was constructed to build "marker-class-activity" topological map in this study. The results showed that the 42 metabolites are mainly involved in lipid metabolism, cardiovascular and nervous system, inflammation and oxidative stress, in addition to some nutrients (Fig. 4D).Table 1 Identification of significantly differential expressed endogenous metabolites in the serum of ratsa.

Table 1No.	Identified potential biomarkers	VIP	Concentration (Intensity/1000) b	F c	Sig. c	p value d	
CON	SMN	
BM01	Valine	1.6218	57.49 ± 13.05	76.04 ± 8.33	3.166	0.092	1.34 × 10−3	
BM02	D-Erythro-imidazole-glycerol-phosphate	1.9156	7.48 ± 1.16	5.77 ± 0.79	1.148	0.298	1.16 × 10−3	
BM03	Arabinofuranobiose	1.7494	56.70 ± 4.83	46.81 ± 5.67	0.754	0.397	5.42 × 10−4	
BM04	9(S)-HODE	1.6464	20.81 ± 3.71	17.32 ± 2.56	1.272	0.274	2.51 × 10−2	
BM05	3-Hydroxyhexadecanoylcarnitine	1.6937	89.94 ± 21.23	117.63 ± 13.15	2.321	0.145	2.52 × 10−3	
BM06	DHA	1.6588	46.66 ± 11.01	61.69 ± 8.32	0.876	0.362	2.90 × 10−3	
BM07	5-methyltetrahydrofolic acid	1.7986	133.57 ± 34.02	183.43 ± 22.2	2.558	0.127	1.09 × 10−3	
BM08	2-Hydroxyestrone-1-S-glutathione	1.6781	252.63 ± 73.23	342.07 ± 46.89	4.166	0.056	4.42 × 10−3	
BM09	3-O-Sulfogalactosylceramide	1.6276	159.97 ± 44.24	209.14 ± 25.68	4.703	0.044	8.57 × 10−3	
BM10	23S,25-dihydroxyvitamin D3	1.8214	25.87 ± 6.97	36.67 ± 5.69	0.030	0.864	1.32 × 10−3	
BM11	13′-Carboxy-alpha-tocopherol	1.7835	38.37 ± 9.95	57.08 ± 12.38	0.468	0.502	1.55 × 10−3	
BM12	Neuromedin N (1–4)	1.6988	38.34 ± 11.20	56.77 ± 12.74	0.097	0.759	2.95 × 10−3	
BM13	Ubiquinol-6	1.7079	39.57 ± 11.34	57.04 ± 13.14	0.466	0.504	5.14 × 10−3	
BM14	Pipecolic acid	1.5759	35.87 ± 9.05	23.56 ± 9.04	0.226	0.640	7.03 × 10−3	
BM15	Indole-3-propionic acid	1.6222	52.74 ± 13.80	33.44 ± 12.72	0.585	0.454	4.43 × 10−3	
BM16	Creatinine	1.5306	41.21 ± 5.67	31.71 ± 2.50	3.632	0.072	1.30 × 10−4	
BM17	Taurocholic acid	1.8613	1679.74 ± 177.20	4656.77 ± 1853.36	10.473	0.005	6.47 × 10−4	
BM18	11′-Carboxy-alpha-tocotrienol	1.8793	93.90 ± 11.55	231.42 ± 75.54	10.068	0.005	2.51 × 10−4	
BM19	LysoPC(16:1)	1.7688	34.49 ± 2.10	29.46 ± 4.86	4.729	0.043	1.08 × 10−2	
BM20	Allantoin	1.8919	340.50 ± 82.05	498.20 ± 39.15	10.542	0.004	1.08 × 10−4	
BM21	TG(24:0)	1.5438	63.66 ± 15.06	82.77 ± 8.95	0.564	0.462	2.86 × 10−3	
BM22	LysoPC(P-16:0)	1.6293	81.97 ± 18.33	100.86 ± 8.58	1.81	0.195	8.54 × 10−3	
BM23	Dihydrothymine	1.7401	23.92 ± 8.33	14.60 ± 3.70	6.666	0.019	6.89 × 10−3	
BM24	LysoPC(18:0)	1.6055	94.30 ± 15.67	70.62 ± 11.95	0.576	0.458	1.31 × 10−3	
BM25	L-Cystine	1.7164	194.62 ± 33.42	144.52 ± 26.54	0.291	0.596	1.60 × 10−3	
BM26	Adrenoyl ethanolamide	1.7359	366.93 ± 48.07	279.79 ± 41.08	0.379	0.546	3.79 × 10−4	
BM27	Deoxycholic acid	1.8089	164.85 ± 22.73	209.20 ± 17.43	0.417	0.526	1.16 × 10−4	
BM28	Tetranor 12-HETE	1.7192	341.23 ± 39.23	277.04 ± 27.13	1.641	0.217	4.76 × 10−4	
BM29	8(R)-Hydroperoxylinoleic acid	1.5482	197.54 ± 47.24	263.72 ± 17.63	9.912	0.006	1.48 × 10-3-	
BM30	LysoPE(22:6)	1.5006	324.37 ± 104.63	187.82 ± 53.81	5.212	0.035	2.68 × 10−3	
BM31	20-Hydroxy-leukotriene E	1.6787	198.64 ± 30.58	138.83 ± 15.69	11.621	0.003	9.03 × 10−5	
BM32	LysoPC(26:1)	1.6427	9.05 ± 2.15	12.63 ± 2.01	0.010	0.920	1.16 × 10−3	
BM33	LysoPE(18:2)	1.5617	20.69 ± 6.37	41.33 ± 14.71	6.108	0.024	1.48 × 10−3	
BM34	LysoPI(18:0)	1.5380	1.89 ± 0.63	2.81 ± 0.72	0.685	0.419	6.87 × 10−3	
BM35	Isoacitretin	1.6593	36.53 ± 4.79	30.14 ± 4.32	0.171	0.684	5.69 × 10−3	
BM36	9-OxoODE	2.0438	4.11 ± 0.98	5.24 ± 0.88	0.005	0.943	1.40 × 10−2	
BM37	5-HETE	1.6259	17.23 ± 2.02	13.10 ± 1.58	0.284	0.600	7.62 × 10−5	
BM38	5-Aminopentanamide	1.6725	37.80 ± 2.64	31.11 ± 1.46	2.574	0.126	1.50 × 10−6	
BM39	Homocysteine thiolactone	2.0244	6.59 ± 0.81	9.38 ± 1.59	15.229	0.001	2.43 × 10−4	
BM40	Leukotriene C5	1.7129	266.23 ± 39.35	316.19 ± 21.94	4.243	0.054	2.52 × 10−3	
BM41	Hypoxanthine	1.5229	101.61 ± 21.24	74.84 ± 15.99	0.861	0.366	5.13 × 10−3	
BM42	Cholesterol sulfate	1.9862	50.40 ± 10.60	16.04 ± 7.80	0.802	0.382	1.57 × 10−7	
a The presence of metabolites was verified using Rt and m/z, compared to genuine chemicals.

b The mean ± standard deviation is used to represent the concentration of potential biomarkers in rat serum.

c The F and Sig. values were derived from the t-test of independent samples. The F value represents the ratio of the variance among the groups to the variance within the group.

d p values were calculated from the t-test (CON: control group; SMN: koumiss group).

Fig. 4 Analysis of potential biomarkers and related pathway. (A) Overview of enriched metabolite sets (top 25); (B) Heat map of 42 potential biomarkers between groups (red, up regulated; green, down regulated). Rows: metabolites; Columns: samples (K: control group; M: koumiss group); (C) Pathway analysis; (D) Network topology on class and bioactivity of potential biomarkers (diamond: potential biomarkers; triangle: class; inverted triangle: bioactivity).

Fig. 4

4 Discussion

4.1 Koumiss contains a variety of nutrients that the body needs

Koumiss is a beverage that is abundant in essential nutrients required by the body, including protein, fat, lactose, vitamins and minerals. Research indicates that koumiss contains higher levels of essential amino acids, unsaturated fatty acids, lactose and vitamins compared to other types of domestic animal milk [14,15]. In this study, metabolomics investigation screened thirteen nutrients or related metabolites among the 42 potential biomarkers: Valine, D-Erythro-imidazole-glycerol-phosphate, Arabinofuranobiose, DHA, 5-methyltetrahydrofolic acid, 2-Hydroxyestrone-1-S-glutathione, 23S,25-dihydroxyvitamin D3, 13′-Carboxy-alpha-tocopherol, Pipecolic acid, Indole-3-propionic acid, 11′-Carboxy-alpha-tocotrienol, L-Cystine and 5-Aminopentanamide.After administration of daily koumiss on normal rats, the contents of amino acids and vitamins, such as Valine, DHA, 5-methyltetrahydrofolicacidand 13′-Carboxy-alpha-tocopherol, increased significantly in serum of murine (Fig. 5). Koumiss plays a positive effect on cardio-cerebrovascular and nervous system. These findings provide straightforward evidence for the high nutritional value of koumiss based on endogenous terminal metabolism.Fig. 5 The underlying mechanism of Koumiss ameliorating Cardio-Cerebrovascular Systems.

Fig. 5

4.2 Koumiss influents inflammation and immune regulatory of rats

Consuming Koumiss on a daily basis has been shown to enhance the immune system, as approximately 80 % of the body's tissues are located in the intestines. The immune system experiences a notable decline following the elimination of intestinal bacteria. Research has demonstrated that animals lacking gut bacteria exhibit reduced levels of crucial white blood cells and protective chemicals in their bloodstream. Upon reintroduction of naturally occurring bacteria into the intestinal tract of these animals, white blood cells are activated, leading to a reinforcement of the immune system. Bacteria present in fermented foods have been found to produce chemicals that can traverse the intestinal barrier and induce the generation of immune cells within the immune system [16]. Research on the impact of Koumiss on the immune system and its support of antibacterial properties has demonstrated significant enhancements in the immune response of test subjects. Consumption of fresh mare milk has been shown to increase the thymus and spleen indices, enhance macrophage function, and elevate hemolysin levels in the blood serum. In addition, koumiss has been shown to enhance the weight of immune organs in BALB/c mice, improve normal immune functions, regulate cell immune capabilities, and modulate abnormal body fluid immune responses [17].

The results of this experiment also suggest that koumiss influents inflammation and immune regulatory of rats. There are five components belonging to arachidonic acids (AA) and linoleic acids (LA) screened as potential biomarkers in this experiment. Arachidonic acid and linoleic acid are implicated immune regulatory, pro-inflammatory, inflammation resolving, platelet aggregation and neuronal signaling [18,19], that are correspondingly related to the pathophysiology of metabolic and cardio-cerebrovascular [20,21]. Tetranor 12-HETE is the major β-oxidation product resulting from peroxisomal metabolism of 12(S)-HETE, which plays a role in inflammation, immune cell recruitment, vasoconstriction, and neurological function [18,22,23]. 20-Hydroxy-leukotriene E4 is a metabolite that can originate from the lipid oxidation of leukotriene E4 (LTE4), which is a kind of cysteinyl leukotriene as potent inflammatory mediators [18,24]. Leukotriene C5 is a slow reacting substance derived from eicosapentaenoic acid and has similar biological activity as leukotriene C4, which is also a kind of cysteinyl leukotriene [25]. 9-OxoODE belongs to the class of organic compounds known as lineolic acids and derivatives, and 5-HETEis an endogenous eicosanoid involved in the pathway of leukotriene synthesis [18]. After koumiss administration, the level of Tetranor 12-HETE, 5-hydroxyeicosatetraenoic acid (5-HETE) and Leukotriene C5 increased and 20-Hydroxy-leukotriene E, 9-OxoODE decreased significantly. The contents of above five metabolites showed different trends.

Research conducted on healthy human adults has shown that higher consumption of AA or LA does not lead to an increase in levels of inflammatory markers. Additionally, epidemiological studies have indicated a potential link between ARA and LA and reduced inflammation [18]. While inflammation is a normal process that is part of host defence and tissue healing [26], the effect of koumiss on inflammation called further targeted researches. Considering the reports on immune regulatory function of koumiss [3], enzyme linked immunosorbent assay was conducted to detect the level of immune cytokines in this work. The results showed that the content of IFN-γ, IgA and IgG in serum increased significantly in SMN group (p < 0.05), confirming the effect of koumiss on enhancing immunity.

4.3 Koumiss regulates lipid metabolism of normal rats

Recent years, there has been a growing emphasis on scientific research regarding natural food products that have the potential to effectively reduce serum cholesterol levels with minimal or no adverse effects. Specifically, the probiotic properties of LAB cultures have been under evaluation. One notable benefit of probiotic-rich foods is their ability to lower serum cholesterol levels [27]. There is a continued interest in the development of bioactive compounds from food sources, such as traditional dairy products or dairy products fortified with Lactobacillus acidophilus like Koumiss, that have cholesterol-lowering properties [28]. Consistent intake of dairy products offers three essential antihypertensive nutrients (calcium, whey-derived peptides, and casein phosphopeptides) as well as additional active peptides that have been shown to reduce blood pressure [29].

After the intervention of koumiss, the contents of the above metabolites in rats were statistically differently, indicating that daily drinking of koumiss can regulate the lipid metabolism of normal healthy body. There are six potential biomarkers involved in lipid metabolism: 3-Hydroxy-hexadecanoylcarnitine, DHA, Ubiquinol-6, Taurocholic acid, Deoxycholic acid and 8(R)-hydroperoxylinoleic acid. Besides, seven lysophospholipids, as a class of functional lipids with high abundance in blood [30], were also screened as potential biomarkers in this work.

3-Hydroxy-hexadecanoylcarnitine is a long chain acylcarnitine that generated from long-chain fatty acids in the diet, and its function is to ensure long-chain fatty acids transport into the mitochondria [31]. It is thought as a feedback inhibition mechanism of insulin resistance [32,33]. The level of long chain acylcarnitine in blood increased when the oxidation of fatty acids is incompletely or the metabolism of carbohydrate and lipid altered [34]. In chronic heart failure patients, long chain acylcarnitine metabolite level was reported to increase in several studies [[35], [36], [37]]. DHA is an omega-3 essential fatty acid. It can reduce the levels of triglycerides in the blood and prevent them from lining the arterial walls, leading to decrease chances of contracting heart diseases considerably [[38], [39], [40]]. Ubiquinol-6 is an effective antioxidant in a number of membrane and biological systems by preventing peroxidative damage to lipids [41,42]. Taurocholic acid, Deoxycholic acid and 8(R)-hydroperoxylinoleic acid belongs to bile acids and oxidative product, playing an important role in lipid metabolism [[43], [44], [45]].

Lysophospholipids, including lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and lysophosphatidylinositol (LPI), have been reported in many metabolomics researches [46]. They are highly abundant in plasma and could bind to protein carriers in the extracellular milieu. The amphiphilic properties of some of them enable their extracellular secretion and significance as signaling molecules [47]. For example, has been recently recognized as a crucial contributor to the biosynthesis of neuronal membranes [48]. It has also been documented to have inflammatory, anti-coagulant, and cytotoxic functions [49]. Moreover, LPC has the ability to enhance oxidative stress and trigger abnormalities in cell structure and function, which eventually lead to hemodynamic disturbances [50,51]. Elevated amounts of LPC might intensify the production of nitric oxide, leading to a rise in the creation of reactive oxidative species (ROS). This could further cause damage to endothelial cells in atherosclerosis and cardiovascular diseases [52,53]. Numerous researches have shown that LPE participates in a variety of cell functions, especially in the differentiation and migration of PC-12 neuronal cells [54]. It was reported as a neurotrophic activator via activation of MAPK cascade, of which induced neuronal differentiation and suppressed serum deprivation-induced apoptosis. LPI is recognized for triggering signaling routes related to cell growth, movement, and tumor formation [55]. Of note, LPI has the ability to act as a lipid signaling molecule, participating in the control of fat accumulation and glucose balance [56], indicating a physiological role of LPI in regulation and metabolism.

4.4 Koumiss has therapeutic potential in cardio-cerebrovascular and nervous system

There are nine potential biomarkers involved in regulating of cardio-cerebrovascular and nervous system: Valine, α-Dimorphecolic acid, 5-methyltetrahydrofolic acid (5-MTHF), 2-Hydroxyestrone-1-S-glutathione, 3-O-Sulfogalactosylceramide, Neuromedin N (1–4), DHA, Hypoxanthine and Cholesterol sulfate. The observed decrease in risk metabolites and increase in metabolites with beneficial regulatory functions suggest that koumiss may have a positive impact on the cardiovascular, cerebrovascular, and nervous systems in healthy individuals.

α-Dimorphecolic acid is synthesized from linoleic acid. It can increase plasminogen activator inhibitor type-1 (PAI-1) expression by activates peroxisomal proliferator-activated receptor-gamma (PPARγ) in human endothelial cells, leading to increases risk for myocardial infarction and venous thrombosis [57,58]. Elevated level of Hypoxanthine is a risk factor for cardiovascular diseases, neurodegenerative diseases, and neural tube defect [59,60]. Cholesterol sulfate is an endogenous steroid and the C3β sulfate ester of cholesterol. In terms of quantity, it is the most significant sterol sulfate identified in human plasma, serving as a part of cell membranes. Although Cholesterol sulfate has neuroprotective effects in brain [61]. It is a potent inhibitor of both plasma thrombin and plasmin in blood which is closely related to thrombosis [62,63]. The results showed that the serum contents of the above three risk metabolites decreased in SMN group.

Valine is one of the essential amino acids for body，and its deficiency is marked by neurological defects in the brain [64,65]. It is one kind of branched chain amino acids (BCAAs), that play critical roles of nutrition physiological functions in glucose and lipid metabolism, protein synthesis, as well as intestinal health and immune [66]. 5-MTHF is a form of external supplementation for folate, whose deficiency has been linked with an increased risk of cardiovascular disease [67], neural tube defects [68], cancer and cognitive dysfunction [69,70]. Compared to folic acid, 5-MTHF can be absorbed well even when gastrointestinal pH is altered and its bioavailability won't be impacted by congenital deficiency of methyl tetrahydrofolate reductase. Besides, 5-MTHF can also prevent the potential negative effects of unconverted folic acid in the peripheral circulation [71,72]. 2-Hydroxyestrone-1-S-glutathioneis a glutathione conjugate derivative of estrogen that is a kind of steroid compound with extensive biological activity in cardiovascular system, endocrine system, body metabolism, bone growth and maturity and skin [[73], [74], [75]]. In addition, 3-O-Sulfogalactosylceramide, Neuromedin N (1–4) and DHA play an important regulatory role in nerve signal transduction, membrane protection, blood pressure and immune regulatory [[76], [77], [78], [79]]. The results showed that the serum contents of the above six metabolites with positive regulatory function increased in SMN group.

5 Conclusion

Koumiss is a conventional fermented item with a rich nutritional value and beneficial features. It has therapeutic efficacy on regulating lipid metabolism and enhancing immunity. This study confirmed the benign regulatory effect of koumiss on normal organism from the perspective of endogenous metabolites, and provided objective support for the promotion and application of this ethnic food.

Funding

This research was funded by Major Science and Technology Projects in Xinjiang Uygur Autonomous Region, grant number 2017A01002 .

Data availability statement

Data available on request from the corresponding author upon reasonable request.

Disclaimer/Publisher's note

The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

CRediT authorship contribution statement

Leqi Wang: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Formal analysis, Data curation. Yuanfang Sun: Software, Investigation, Data curation. Lijing Du: Methodology, Formal analysis, Data curation. Qian Wang: Investigation. Min Zhan: Validation. Shasha Li: Writing – review & editing, Supervision, Resources. Xue Xiao: Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xue Xiao reports financial support was provided by Major Science and Technology Projects in Xinjiang Uygur Autonomous Region. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

All authors thank Jun Luo for the information of Koumiss, and the members of Xinjiang Xinziyuan Biopharmaceutical Co., Ltd for the help in supplying the Koumiss.
==== Refs
References

1 Afzaal M. Saeed F. Anjum F. Waris N. Husaain M. Ikram A. Ateeq H. Muhammad Anjum F. Suleria H. Nutritional and ethnomedicinal scenario of koumiss: a concurrent review Food Sci. Nutr. 9 2021 6421 6428 10.1002/fsn3.2595 34760271
2 Petrova P. Ivanov I. Tsigoriyna L. Valcheva N. Vasileva E. Parvanova-Mancheva T. Arsov A. Petrov K. Traditional Bulgarian dairy products: ethnic foods with health benefits Microorganisms 9 2021 10.3390/microorganisms9030480
3 Li Q. Zhang C. Xilin T. Ji M. Meng X. Zhao Y. Siqin B. Zhang N. Li M. Effects of koumiss on intestinal immune modulation in immunosuppressed rats Front. Nutr. 9 2022 765499 10.3389/fnut.2022.765499
4 Chen Y. Wang Z. Chen X. Liu Y. Zhang H. Sun T. Identification of angiotensin I-converting enzyme inhibitory peptides from koumiss, a traditional fermented mare's milk J. Dairy Sci. 93 2010 884 892 10.3168/jds.2009-2672 20172208
5 Ren S. Chen A. Tian Y. Bai Z. Wang C. Lactobacillus paracasei from koumiss ameliorates diarrhea in mice via tight junctions modulation Nutrition 98 2022 111584 10.1016/j.nut.2021.111584
6 Wu Y. Li Y. Gesudu Q. Zhang J. Sun Z. Halatu H. Menghe B. Liu W. Bacterial composition and function during fermentation of Mongolia koumiss Food Sci. Nutr. 9 2021 4146 4155 10.1002/fsn3.2377 34401066
7 Bai M. Qing M. Guo Z. Zhang Y. Chen X. Bao Q. Zhang H. Sun T.S. Occurrence and dominance of yeast species in naturally fermented milk from the Tibetan Plateau of China Can. J. Microbiol. 56 2010 707 714 10.1139/w10-056 20921981
8 Guo L. Ya M. Guo Y.S. Xu W.L. Li C.D. Sun J.P. Zhu J.J. Qian J.P. Study of bacterial and fungal community structures in traditional koumiss from Inner Mongolia J. Dairy Sci. 102 2019 1972 1984 10.3168/jds.2018-15155 30639001
9 Mu Z. Yang X. Yuan H. Detection and identification of wild yeast in Koumiss Food microbiology 31 2012 301 308 10.1016/j.fm.2012.04.004 22608237
10 Rakhmanova A. Wang T. Xing G. Ma L. Hong Y. Lu Y. Xin L. Xin W. Zhu Q. Lu X. Isolation and identification of microorganisms in Kazakhstan koumiss and their application in preparing cow-milk koumiss J. Dairy Sci. 104 2021 151 166 10.3168/jds.2020-18527 33162079
11 Li S. Zhan M. Yan S. Xiao X. The antifatigue mechanism of buyang-huanwu decoction as revealed by serum metabolomics in an endurance swimming rat model J. Med. Food 25 11 2022 1038 1049 10.1089/jmf.2022.K.0014 36448732
12 Li S. Xiao X. Han L. Wang Y. Luo G. Renoprotective effect of Zhenwu decoction against renal fibrosis by regulation of oxidative damage and energy metabolism disorder Sci. Rep. 8 1 2018 14627 10.1038/s41598-018-32115-9
13 Du L. Sun Y. Wang Q. Wang L. Zhang Y. Li S. Jin H. Yan S. Xiao X. Integrated metabolomics and 16S rDNA sequencing to investigate the mechanism of immune-enhancing effect of health Tonic oral liquid Food Res. Int. 144 2021 110323 10.1016/j.foodres.2021.110323
14 Pastuszka R. Barlowska J. Litwinczuk Z. Allergenicity of milk of different animal species in relation to human milk Postepy Hig. Med. Dosw. 70 2016 1451 1459 10.5604/17322693.1227842
15 Barreto I. Urbano S.A. Oliveira C.A.A. Macedo C.S. Borba L.H.F. Chags B.M.E. Rangel A.H.N. Chemical composition and lipid profile of mare colostrum and milk of the quarter horse breed PLoS One 15 2020 e0238921 10.1371/journal.pone.0238921
16 Wang H. Hussain T. Yao J. Li J. Sabir N. Liao Y. Liang Z. Wang Y. Liu Y. Zhao D. Zhou X. Koumiss promotes Mycobacterium bovis infection by disturbing intestinal flora and inhibiting endoplasmic reticulum stress Faseb. J. 35 9 2021 e21777 10.1096/fj.202002485RR official publication of the Federation of American Societies for Experimental Biology
17 Ya T. Zhang Q. Chu F. Merritt J. Bilige M. Sun T. Du R. Zhang H. Immunological evaluation of Lactobacillus casei Zhang: a newly isolated strain from koumiss in Inner Mongolia China. BMC immunology 9 2008 68 10.1186/1471-2172-9-68 19019236
18 Sonnweber T. Pizzini A. Nairz M. Weiss G. Tancevski I. Arachidonic acid metabolites in cardiovascular and metabolic diseases Int. J. Mol. Sci. 19 2018 10.3390/ijms19113285
19 Innes J.K. Calder P.C. Omega-6 fatty acids and inflammation Prostaglandins, leukotrienes, and essential fatty acids 132 2018 41 48 10.1016/j.plefa.2018.03.004 29610056
20 Mitchell J.A. Kirkby N.S. Eicosanoids, prostacyclin and cyclooxygenase in the cardiovascular system Br. J. Pharmacol. 176 2019 1038 1050 10.1111/bph.14167 29468666
21 Zhou Y. Khan H. Xiao J. Cheang W.S. Effects of arachidonic acid metabolites on cardiovascular health and disease Int. J. Mol. Sci. 22 2021 10.3390/ijms222112029
22 Gonzalez-Nunez D. Claria J. Rivera F. Poch E. Increased levels of 12(S)-HETE in patients with essential hypertension Hypertension 37 2001 334 338 10.1161/01.hyp.37.2.334 11230294
23 Zheng Z. Li Y. Jin G. Huang T. Zou M. Duan S. The biological role of arachidonic acid 12-lipoxygenase (ALOX12) in various human diseases Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 129 2020 110354 10.1016/j.biopha.2020.110354
24 Austen K.F. Maekawa A. Kanaoka Y. Boyce J.A. The leukotriene E4 puzzle: finding the missing pieces and revealing the pathobiologic implications The Journal of allergy and clinical immunology 124 2009 406 414 10.1016/j.jaci.2009.05.046 quiz 415-406 19647860
25 Hammarstrom S. Leukotriene C5: a slow reacting substance derived from eicosapentaenoic acid J. Biol. Chem. 255 1980 7093 7094 6104669
26 Medzhitov R. Origin and physiological roles of inflammation Nature 454 2008 428 435 10.1038/nature07201 18650913
27 Lye H.S. Rusul G. Liong M.T. Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol J. Dairy Sci. 93 4 2010 1383 1392 10.3168/jds.2009-2574 20338415
28 Pan D.D. Zeng X.Q. Yan Y.T. Characterisation of Lactobacillus fermentum SM-7 isolated from koumiss, a potential probiotic bacterium with cholesterol-lowering effects J. Sci. Food Agric. 91 3 2011 512 518 10.1002/jsfa.4214 21218486
29 Afzaal M. Saeed F. Anjum F. Waris N. Husaain M. Ikram A. Ateeq H. Muhammad Anjum F. Suleria H. Nutritional and ethnomedicinal scenario of koumiss: a concurrent review Food Sci. Nutr. 9 11 2021 6421 6428 10.1002/fsn3.2595 34760271
30 Quehenberger O. Armando A.M. Brown A.H. Milne S.B. Myers D.S. Merrill A.H. Bandyopadhyay S. Jones K.N. Kelly S. Shaner R.L. Lipidomics reveals a remarkable diversity of lipids in human plasma Journal of lipid research 51 2010 3299 3305 10.1194/jlr.M009449 20671299
31 Gnoni A. Longo S. Gnoni G.V. Giudetti A.M. Carnitine in human muscle bioenergetics: can carnitine supplementation improve physical exercise? Molecules 25 2020 10.3390/molecules25010182
32 Schooneman M.G. Vaz F.M. Houten S.M. Soeters M.R. Acylcarnitines: reflecting or inflicting insulin resistance? Diabetes 62 2013 1 8 10.2337/db12-0466 23258903
33 Vilks K. Videja M. Makrecka-Kuka M. Katkevics M. Sevostjanovs E. Grandane A. Dambrova M. Liepinsh E. Long-chain acylcarnitines decrease the phosphorylation of the insulin receptor at Tyr1151 through a PTP1B-dependent mechanism Int. J. Mol. Sci. 22 2021 10.3390/ijms22126470
34 Vissing C.R. Duno M. Wibrand F. Christensen M. Vissing J. Hydroxylated long-chain acylcarnitines are biomarkers of mitochondrial myopathy The Journal of clinical endocrinology and metabolism 104 2019 5968 5976 10.1210/jc.2019-00721 31294795
35 Ahmad T. Kelly J.P. McGarrah R.W. Hellkamp A.S. Fiuzat M. Testani J.M. Wang T.S. Verma A. Samsky M.D. Donahue M.P. Prognostic implications of long-chain acylcarnitines in heart failure and reversibility with mechanical circulatory support J. Am. Coll. Cardiol. 67 2016 291 299 10.1016/j.jacc.2015.10.079 26796394
36 Liepinsh E. Kuka J. Vilks K. Svalbe B. Stelfa G. Vilskersts R. Sevostjanovs E. Goldins N.R. Groma V. Grinberga S. Low cardiac content of long-chain acylcarnitines in TMLHE knockout mice prevents ischaemia-reperfusion-induced mitochondrial and cardiac damage Free radical biology & medicine 177 2021 370 380 10.1016/j.freeradbiomed.2021.10.035 34728372
37 Liepinsh E. Makrecka-Kuka M. Volska K. Kuka J. Makarova E. Antone U. Sevostjanovs E. Vilskersts R. Strods A. Tars K. Long-chain acylcarnitines determine ischaemia/reperfusion-induced damage in heart mitochondria The Biochemical journal 473 2016 1191 1202 10.1042/BCJ20160164 26936967
38 Horrocks L.A. Yeo Y.K. Health benefits of docosahexaenoic acid (DHA) Pharmacol. Res. 40 1999 211 225 10.1006/phrs.1999.0495 10479465
39 Li J. Pora B.L.R. Dong K. Hasjim J. Health benefits of docosahexaenoic acid and its bioavailability: a review Food Sci. Nutr. 9 2021 5229 5243 10.1002/fsn3.2299 34532031
40 Siriwardhana N. Kalupahana N.S. Moustaid-Moussa N. Health benefits of n-3 polyunsaturated fatty acids: eicosapentaenoic acid and docosahexaenoic acid Adv. Food Nutr. Res. 65 2012 211 222 10.1016/B978-0-12-416003-3.00013-5 22361189
41 Schultz J.R. Ellerby L.M. Gralla E.B. Valentine J.S. Clarke C.F. Autoxidation of ubiquinol-6 is independent of superoxide dismutase Biochemistry 35 1996 6595 6603 10.1021/bi960245h 8639607
42 Lokhmatikov A.V. Voskoboynikova N. Cherepanov D.A. Skulachev M.V. Steinhoff H.J. Skulachev V.P. Mulkidjanian A.Y. Impact of antioxidants on cardiolipin oxidation in liposomes: why mitochondrial cardiolipin serves as an apoptotic signal? Oxid. Med. Cell. Longev. 2016 2016 8679469 10.1155/2016/8679469
43 Xu J. Xie S. Chi S. Zhang S. Cao J. Tan B. Protective effects of taurocholic acid on excessive hepatic lipid accumulation via regulation of bile acid metabolism in grouper Food Funct. 13 2022 3050 3062 10.1039/d1fo04085e 35199809
44 Qi Y. Jiang C. Cheng J. Krausz K.W. Li T. Ferrell J.M. Gonzalez F.J. Chiang J.Y. Bile acid signaling in lipid metabolism: metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice Biochim. Biophys. Acta 1851 2015 19 29 10.1016/j.bbalip.2014.04.008 24796972
45 LaRusso N.F. Szczepanik P.A. Hofmann A.F. Effect of deoxycholic acid ingestion on bile acid metabolism and biliary lipid secretion in normal subjects Gastroenterology 72 1977 132 140 318580
46 Tan S.T. Ramesh T. Toh X.R. Nguyen L.N. Emerging roles of lysophospholipids in health and disease Prog. Lipid Res. 80 2020 101068 10.1016/j.plipres.2020.101068
47 Xu Y. Xiao Y.J. Baudhuin L.M. Schwartz B.M. The role and clinical applications of bioactive lysolipids in ovarian cancer J. Soc. Gynecol. Invest. 8 2001 1 13
48 Wong B.H. Chan J.P. Cazenave-Gassiot A. Poh R.W. Foo J.C. Galam D.L. Ghosh S. Nguyen L.N. Barathi V.A. Yeo S.W. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid (DHA) in eye and is important for photoreceptor cell development J. Biol. Chem. 291 2016 10501 10514 10.1074/jbc.M116.721340 27008858
49 Plemel J.R. Michaels N.J. Weishaupt N. Caprariello A.V. Keough M.B. Rogers J.A. Yukseloglu A. Lim J. Patel V.V. Rawji K.S. Mechanisms of lysophosphatidylcholine-induced demyelination: a primary lipid disrupting myelinopathy Glia 66 2018 327 347 10.1002/glia.23245 29068088
50 Chen H. Cao G. Chen D.Q. Wang M. Vaziri N.D. Zhang Z.H. Mao J.R. Bai X. Zhao Y.Y. Metabolomics insights into activated redox signaling and lipid metabolism dysfunction in chronic kidney disease progression Redox Biol. 10 2016 168 178 10.1016/j.redox.2016.09.014 27750081
51 Yasunari K. Maeda K. Minami M. Yoshikawa J. HMG-CoA reductase inhibitors prevent migration of human coronary smooth muscle cells through suppression of increase in oxidative stress Arterioscler. Thromb. Vasc. Biol. 21 2001 937 942 10.1161/01.atv.21.6.937 11397700
52 Kim E.A. Kim J.A. Park M.H. Jung S.C. Suh S.H. Pang M.G. Kim Y.J. Lysophosphatidylcholine induces endothelial cell injury by nitric oxide production through oxidative stress J. Matern. Fetal Neonatal Med. : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet 22 2009 325 331 10.1080/14767050802556075
53 Schmitz G. Ruebsaamen K. Metabolism and atherogenic disease association of lysophosphatidylcholine Atherosclerosis 208 2010 10 18 10.1016/j.atherosclerosis.2009.05.029 19570538
54 Nishina A. Kimura H. Sekiguchi A. Fukumoto R.H. Nakajima S. Furukawa S. Lysophosphatidylethanolamine in Grifola frondosa as a neurotrophic activator via activation of MAPK Journal of lipid research 47 2006 1434 1443 10.1194/jlr.M600045-JLR200 16614393
55 Ruban E.L. Ferro R. Arifin S.A. Falasca M. Lysophosphatidylinositol: a novel link between ABC transporters and G-protein-coupled receptors Biochem. Soc. Trans. 42 2014 1372 1377 10.1042/Bst20140151 25233417
56 Arifin S.A. Falasca M. Lysophosphatidylinositol signalling and metabolic diseases Metabolites 6 2016 10.3390/metabo6010006
57 Marx N. Bourcier T. Sukhova G.K. Libby P. Plutzky J. PPARgamma activation in human endothelial cells increases plasminogen activator inhibitor type-1 expression: PPARgamma as a potential mediator in vascular disease Arterioscler. Thromb. Vasc. Biol. 19 1999 546 551 10.1161/01.atv.19.3.546 10073956
58 Hampel J.K.A. Brownrigg L.M. Vignarajah D. Croft K.D. Dharmarajan A.M. Bentel J.M. Puddey I.B. Yeap B.B. Differential modulation of cell cycle, apoptosis and PPAR gamma 2 gene expression by PPAR gamma agonists ciglitazone and 9-hydroxyoctadecadienoic acid in monocytic cells Prostag Leukotr Ess 74 2006 283 293 10.1016/j.plefa.2006.03.002
59 Ryu H.M. Kim Y.J. Oh E.J. Oh S.H. Choi J.Y. Cho J.H. Kim C.D. Park S.H. Kim Y.L. Hypoxanthine induces cholesterol accumulation and incites atherosclerosis in apolipoprotein E-deficient mice and cells J. Cell Mol. Med. 20 2016 2160 2172 10.1111/jcmm.12916 27396856
60 Kundu D. Dubey V.K. Purines and pyrimidines: metabolism, function and potential as therapeutic options in neurodegenerative diseases Current protein & peptide science 21 2021 170 189 10.2174/1389203721999201208200605
61 Prah J. Winters A. Chaudhari K. Hersh J. Liu R. Yang S.H. Cholesterol sulfate alters astrocyte metabolism and provides protection against oxidative stress Brain Res. 1723 2019 146378 10.1016/j.brainres.2019.146378
62 Strott C.A. Higashi Y. Cholesterol sulfate in human physiology: what's it all about? Journal of lipid research 44 2003 1268 1278 10.1194/jlr.R300005-JLR200 12730293
63 Iwamori M. Iwamori Y. Ito N. Regulation of the activities of thrombin and plasmin by cholesterol sulfate as a physiological inhibitor in human plasma Journal of biochemistry 125 1999 594 601 10.1093/oxfordjournals.jbchem.a022325 10050049
64 Wang J. Liang X.F. He S. Zhang Y.P. Li J. Huang K. Shi L.J. Ren P. Valine acts as a nutritional signal in brain to activate TORC1 and attenuate postprandial ammonia-N excretion in Chinese perch (Siniperca chuatsi) Fish Physiol. Biochem. 46 2020 2015 2025 10.1007/s10695-020-00767-y 32749664
65 Sperringer J.E. Addington A. Hutson S.M. Branched-chain amino acids and brain metabolism Neurochem. Res. 42 2017 1697 1709 10.1007/s11064-017-2261-5 28417264
66 Nie C. He T. Zhang W. Zhang G. Ma X. Branched chain amino acids: beyond nutrition metabolism Int. J. Mol. Sci. 19 2018 10.3390/ijms19040954
67 Li Y. Huang T. Zheng Y. Muka T. Troup J. Hu F.B. Folic acid supplementation and the risk of cardiovascular diseases: a meta-analysis of randomized controlled trials J. Am. Heart Assoc. 5 2016 10.1161/JAHA.116.003768
68 van Gool J.D. Hirche H. Lax H. De Schaepdrijver L. Folic acid and primary prevention of neural tube defects: a review Reprod. Toxicol. 80 2018 73 84 10.1016/j.reprotox.2018.05.004 29777755
69 Pieroth R. Paver S. Day S. Lammersfeld C. Folate and its impact on cancer risk Current nutrition reports 7 2018 70 84 10.1007/s13668-018-0237-y 30099693
70 Ebara S. Nutritional role of folate Congenital. Anom. 57 2017 138 141 10.1111/cga.12233
71 Scaglione F. Panzavolta G. Folate, folic acid and 5-methyltetrahydrofolate are not the same thing Xenobiotica; the fate of foreign compounds in biological systems 44 2014 480 488 10.3109/00498254.2013.845705 24494987
72 Ferrazzi E. Tiso G. Di Martino D. Folic acid versus 5- methyl tetrahydrofolate supplementation in pregnancy Eur. J. Obstet. Gynecol. Reprod. Biol. 253 2020 312 319 10.1016/j.ejogrb.2020.06.012 32868164
73 Patel S. Homaei A. Raju A.B. Meher B.R. Estrogen: the necessary evil for human health, and ways to tame it Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 102 2018 403 411 10.1016/j.biopha.2018.03.078 29573619
74 Knowlton A.A. Lee A.R. Estrogen and the cardiovascular system Pharmacology & therapeutics 135 2012 54 70 10.1016/j.pharmthera.2012.03.007 22484805
75 Raftogianis R. Creveling C. Weinshilboum R. Weisz J. Estrogen metabolism by conjugation J. Natl. Cancer Inst. Monogr. 10 2000 113 124 10.1093/oxfordjournals.jncimonographs.a024234
76 Grassi S. Prioni S. Cabitta L. Aureli M. Sonnino S. Prinetti A. The role of 3-O-sulfogalactosylceramide, sulfatide, in the lateral organization of myelin membrane Neurochem. Res. 41 2016 130 143 10.1007/s11064-015-1747-2 26542149
77 Takahashi T. Suzuki T. Role of sulfatide in normal and pathological cells and tissues Journal of lipid research 53 2012 1437 1450 10.1194/jlr.R026682 22619219
78 Nicoli C.D. Long D.L. Plante T.B. Howard G. Judd S.E. Schulte J. Cushman M. Pro-neurotensin/Neuromedin N and hypertension risk: a prospective study American journal of hypertension 35 2022 281 288 10.1093/ajh/hpab166 34655288
79 Piomelli D. Scalvini L. Fotio Y. Lodola A. Spadoni G. Tarzia G. Mor M. N-acylethanolamine acid amidase (NAAA): structure, function, and inhibition J. Med. Chem. 63 2020 7475 7490 10.1021/acs.jmedchem.0c00191 32191459
