1 Introduction
Lipid is an important component of biological system, which has many important physiological functions in organisms, such as forming cell barrier and membrane matrix, participating in intercellular signal transduction, storing energy and so on. The composition of lipids is highly complex and dynamic, and lipids can be divided into several classes and subclasses according to the differences in polar head groups and linked fatty chains in their structure
[1]. At present, hundreds of thousands of lipid molecules are known, and their content levels are between amol/L and nmol/L. At present, with the development of lipidomics research technology and the deepening of biological research, some new lipid molecules with important physiological functions have been found one after another.
In 2014, Yore et al. compared the lipid changes in the adipose tissue of transgenic mice (AG4OX) overexpressing glucose transporter 4 (Glut4) with those of wild-type mice (WT), and found that there was a kind of molecule in the adipose tissue of AG4OX mice, and its content was about 16-18 times higher than that of WT mice
[2]. After analysis and identification, the molecules were identified as a new class of lipid molecules, whose chemical structure was composed of a fatty acid (FA) and a hydroxy fatty acid (HFA) linked by ester bonds. They named it branched fatty acid esters of hydroxy fatty acids (FAHFAs). Among them, different FA and HFA can form different FAHFAs families, and each FAHFA family can form ester position isomers according to different ester positions
[2]. For example, a compound formed by connecting palmitic acid (PA) and hydroxystearic acid (HSA) through an ester bond is palmitic acid-hydroxy stearic acid (PAHSA); When PA is combined with HSA isomers with different hydroxyl positions to form PAHSA, multiple ester position isomers will be produced, such as 3-PAHSA, 5-PAHSA and 9-PAHSA. In addition, by studying the biological functions of FAHFAs, Yore et al. Found that FAHFAs can improve glucose tolerance, promote the secretion of glucagon-like peptide-1 (GLP-1) and insulin, enhance insulin-stimulated glucose transport and reduce the production of inflammatory factors in immune macrophages in mammals
[2]. Therefore, FAHFAs have anti-T2D and anti-inflammatory effects in mammals, and are expected to be used as a drug for the treatment of T2D
[2⇓~4].
In view of the important biological functions of FAHFAs, they have attracted the attention of chemists and biologists since their emergence. In this paper, we summarized the main research of FAHFAs since its discovery, focusing on the physiological function, metabolism, storage form in vivo, analytical techniques and chemical synthesis of FAHFAs, in order to provide some reference for FAHFAs research.
2 Main research and development of FAHFAs
2.1 Physiological functions of FAHFAs
As mentioned above, Yore et al. First discovered FAHFA and studied its physiological function in 2014 (Figure 1)
[2]. They found that PAHSAs levels were highly correlated with insulin sensitivity and were significantly reduced in adipose tissue and serum in insulin-resistant individuals. PAHSAs can activate G protein-coupled receptor 120 (GPR120) and promote the transport of glucose receptor GLUT-4, thereby enhancing insulin-stimulated glucose uptake. Administration of PAHSAs to mice decreased blood glucose, improved glucose tolerance, and stimulated GLP-1 and insulin secretion. In addition, GPR120 activated by PAHSAs can reduce the production of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in immune macrophages, thus alleviating obesity-related inflammation. These results suggest that PAHSAs play an important role in maintaining glucose homeostasis. Studies on the levels and functions of these lipid molecules may provide new insights into the pathogenesis and treatment of metabolic and inflammatory diseases.
Yore et al. Suggested that FAHFAs are closely related to chronic diseases. Based on this, the current research on the biological function of FAHFAs is mainly to explore the relationship between FAHFAs and a variety of chronic diseases. According to the types of diseases, we briefly introduce the main studies on the physiological functions of FAHFAs from three aspects: FAHFAs and inflammation, FAHFAs and diabetes, and other physiological functions of FAHFAs.
2.1.1 FAHFAs and inflammation
The anti-inflammatory activity of the molecule is particularly important because insulin resistance and the development of T2D are both strongly associated with chronic low-grade inflammation. The first report about FAHFAs and inflammation came from the study of Yore et al. By measuring the levels of TNF-α and IL-1β in ATMs of mice fed with 9-PAHSA for 3 days, they found that the expression of TNF-α and IL-1β in ATMs decreased in mice fed with high-fat diet, indicating that 9-PAHSA has the effect of alleviating obesity-related inflammation
[2]. Since then, some research groups have also shared the research results on the anti-inflammatory activity of FAHFAs
[5⇓~7].
In 2016, Lee et al. Investigated the effects of 5-PAHSA and 9-PAHSA on the treatment of ulcerative colitis by gavage to mice with chronic colitis
[5]. They found that 5-PAHSA and 9-PAHSA reduced body weight, T lymphocyte activity, and secretion of pro-inflammatory and chemokines in mice. These results suggest that PAHSAs may be useful in the treatment of chronic colitis by modulating the intestinal immune system. In the same year, Kuda et al. Found a new anti-inflammatory FAHFA molecule, docosahexaenoic acid-13-hydroxy linoleic acid (13-DHAHLA), in macrophages stimulated by lipopolysaccharide (LPS)
[6]. It can reduce the activation of macrophages induced by LPS and enhance the phagocytosis of zymosan particles, thus achieving anti-inflammatory and inflammatory regression. Recently, Kolar et al. Found that oat oil fractions are rich in FAHFAs and observed that oat oil fractions exhibit anti-inflammatory activity in LPS-stimulated cytokine secretion experiments
[7]. To this end, they identified three LAHLAs that are abundant in oat oil, namely 15-LAHLA, 13-LAHLA and 9-LAHLA, of which 13-LAHLA is the most abundant. Then, they further investigated the anti-inflammatory activity of 13-LAHLA and found that 13-LAHLA could inhibit LPS-stimulated cytokine secretion and proinflammatory gene expression, and showed stronger anti-inflammatory activity than 9-PAHSA.
2.1.2 FAHFAs and diabetes mellitus
On the basis of Yore's research, scientists have carried out more in-depth research on the anti-diabetic properties of FAHFAs. In 2016, Saghatelian and Kahn took PAHSAs as the object to study their function in anti-T2D anti-inflammatory
[8]. They found that PAHSAs levels were highly correlated with insulin sensitivity. Levels of PAHSAs were lower in people with insulin resistance than in healthy people. The intake of PAHSAs can effectively enhance insulin sensitivity and promote insulin secretion. These results suggest that PAHSAs may be involved in the pathogenesis of T2D and may become a new biomarker for measuring insulin resistance and risk of T2D. Subsequently, by studying the therapeutic effect of PAHSAs on T2D, it was found that long-term treatment with PAHSAs could significantly improve the symptoms of dysglycemia in mice fed with high-fat diet (HFD). In the treated HFD mice, the levels of PAHSAs in serum were significantly increased, insulin sensitivity and glucose tolerance were significantly improved, and the levels of inflammatory factors in subcutaneous white fat were also decreased. In addition, the study also found that another important G-protein-coupled receptor, GPR40, is also a target of PAHSAs. This study further confirms the anti-T2D function of FAHFAs and provides new ideas for the treatment of T2D in clinical medicine. Safe insulin sensitizers can be used to prevent and treat insulin resistance, T2D and cardiovascular complications. Except for thiazolidinediones, there are no diabetes drugs mainly used to improve insulin sensitivity on the market at present, and safe insulin sensitizers are expected to make up for the gap in medical needs, and FAHFAs may be a candidate for such drugs
[8,9].
In addition, scientists have also studied the relationship between FAHFAs and insulin-dependent diabetes mellitus (type 1 diabetes mellitus, T1D). Kahn et al. Found that daily administration of PAHSAs to non-obese diabetic (NOD) mice significantly reduced the incidence of T1D and delayed the onset of T1D
[10,11]. This is because PAHSAs can directly protect islet β cells and maintain the survival and function of β cells by weakening the autoimmune response. In the same year, Hamad et al. Reported similar results
[12]. PAHSAs have therapeutic potential for individuals at risk for T1D, given the current lack of effective T1D therapies in the clinic and the few known side effects of PAHSAs.
2.1.3 Studies on Other Physiological Functions of FAHFAs
In 2018, Brezinova et al. First studied FAHFAs in breast milk
[13]. They detected a variety of PAHSAs in breast milk, and the level of 5-PAHSA in breast milk was significantly higher than that in serum and other tissues. In addition, they found that the concentration of PAHSAs in breast milk was related to the degree of obesity of mothers, that is, the concentration of PAHSAs in breast milk of thin mothers was higher than that of obese mothers. The conclusion of the mouse breast milk experiment is also consistent with this feature. FAHFAs can be absorbed by the mother's gastrointestinal tract and then transferred to the newborn, while the effect of 5-PAHSA, which is abundant in breast milk, on neonatal development remains to be determined.
In 2019, Balsinde et al. First explored the relationship between PAHSAs and colorectal cancer by analyzing the changes of 9-hydroxystearic acid (9-HSA) and PAHSAs in human colorectal tumors
[14]. They observed decreased levels of 9-HSA and increased levels of PAHSAs in colorectal tumor tissue compared with paracancerous tissue. Whereas 9-HSA may be a synthetic precursor to 9-PAHSA and has been shown to induce cell cycle arrest and apoptosis. Therefore, they conducted cell experiments to investigate the effects of 9-PAHSA and 9-HSA on colorectal cancer cells (HT-29 and HCT-116). They observed the apoptosis of colorectal cancer cells by adding 9-PAHSA and 9-HSA, respectively, and found that 9-PAHSA did not cause apoptosis of colorectal cancer cells in a wide range of concentrations, while 9-HSA could enhance the synthesis of 9-PAHSA before apoptosis. Therefore, they speculated that the synthesis of PAHSAs in colorectal cancer cells was similar to "a buffer system" and showed a protective effect on the apoptosis of colorectal cancer cells. These results were recently supported by a study by Liu et al., who found elevated levels of PAHLAs in the plasma of patients with colorectal cancer
[15]. In 2017, our research group also made a preliminary exploration on the relationship between FAHFAs and cancer. We analyzed the serum levels of 16 FAHFAs in healthy subjects and breast cancer patients, and observed that the serum levels of 9-PAHSA, 13-PAHSA, 12-SAHSA and 13-SAHSA in breast cancer patients were significantly lower than those in healthy controls
[16]. This suggests that there may be a potential link between FAHFAs and breast cancer, but the specific mechanism needs to be further explored.
Recently, our group analyzed visceral adipose tissue samples from 80 C57BL/6J male mice at nine different months of age (1, 2, 3, 6, 9, 12, 15, 18 and 24 months) to study the relationship between FAHFAs and age
[17]. We observed that the types of FAHFAs in the visceral fat of mice increased with age, and the number of FAHFAs families detected increased from 35 families in infancy (1 month old) to 46 families in old age (18 months old). In addition, we also found that the level of FAHFAs in visceral adipose tissue of mice was highly correlated with age. The mass concentration of FAHFAs in middle age (3 ~ 15 months) was generally lower than that in young age (1 ~ 2 months) and old age (18 ~ 24 months). However, because the visceral fat mass of middle-aged mice is much larger than that of young or old mice, the total amount of FAHFAs in the visceral fat of middle-aged mice is higher. This study provides the metabolic footprint of FAHFAs in mouse visceral fat throughout the life cycle, which is helpful for the study of the mechanism of FAHFAs in growth, development and aging.
Since the initial discovery of anti-T2D and anti-inflammatory functions of FAHFAs, the research on the physiological functions of FAHFAs has gradually expanded. At present, the relationship between chronic diseases or physiological States such as cancer, obesity and aging has been explored, and preliminary results have been achieved. These preliminary results suggest that FAHFAs in vivo may be related to a variety of diseases or physiological States. Unfortunately, at present, most of these studies remain in the observation of preliminary phenomena, and the mechanism of action is not clear, so deeper research needs to be carried out.
2.2 Metabolism of FAHFAs
The study of FAHFAs metabolism is the key to reveal the physiological mechanism of FAHFAs. However, the metabolic mechanism of FAHFAs is still poorly understood, and the synthetic enzymes of FAHFAs have not yet been reported. So far, three kinds of FAHFAs hydrolases have been found.
Parsons et al. Found that two enzymes, androgen induced gene 1 protein (AIG1) and androgen dependent TFPI regulating protein (ADTRP), can specifically hydrolyze FAHFAs through activity-based protein profiling
[18]. The two hydrolases are regioselective, and the farther the ester bond in the FAHFAs structure is from the carboxylic acid group, the faster the hydrolysis rate is. On the contrary, the slower it is. Subsequently, Kolar et al reported a third FAHFAs specific hydrolase, carboxyl ester lipase (CEL)
[19]. CEL, which is present on the pancreatic cell membrane, hydrolyzes FAHFAs at about twice the rate of AIG1 and ADTRP, and has a regioselectivity similar to that of AIG1 and ADTRP.
For the synthesis of FAHFAs in mammals, Kuda et al. Proposed a possible synthesis pathway
[20]. This pathway is related to the antioxidant pathway regulated by nuclear factors Nrf2 and PRDX6. Taking 9-PAHSA as an example, they speculate that the synthesis pathway is as follows: first, the membrane phospholipid (PhL) is oxidized by active oxygen to form phospholipid peroxide (PhL-OOH); Subsequently, PhL-OOH was further converted into phospholipids with hydroxyl fatty acids (PhL-OH) by peroxidase catalysis. Finally, HSA is released from PhL-OH and 9-PAHSA is synthesized under the action of acyl-CoA (Fig. 2). Theoretically, this synthetic route tends to give priority to the synthesis of isomers with ester bonds in the middle of the hydroxyfatty acid chain, while the synthesis mechanism of isomers with ester bonds at both ends of the hydroxyfatty acid chain needs to be studied.
The molecular diversity of FAHFAs makes the anabolic and catabolic pathways of FAHFAs more complex. The study of FAHFAs metabolism is a key issue and an important difficulty in the field of FAHFAs research, and the discovery and determination of its synthetase, hydrolase and metabolic pathway require the cooperation of biologists and chemists.
2.3 Intracellular storage of FAHFAs
In vivo, FAHFAs exist not only in free form but also in bound form. McLean et al. First observed FAHFAs in triglycerides (TAG)
[21]. They found about 150 possible FAHFA-TAGs by analyzing lipids in the paracrimal glands of the hair-tailed possum, a marsupial native to Australia. Remarkably, they did not detect FAHFA-TAGs in adipose tissue elsewhere, such as in the abdomen and around the kidneys, so they speculated that these FAHFA-TAGs in the paracrimal glands might be a signaling molecule that helps animals communicate with each other through smell. This work is the first time to find the existence of FAHFA-TAG, but its chemical structure has not been accurately identified.
Until 2019, Saghatelian et al. Synthesized FAHFA-TAGs standard by chemical synthesis and studied its mass spectrum fragmentation law (Figure 3)
[22]. They detected and identified FAHFA-TAGs in mouse adipose tissue and found that the content of FAHFA-TAGs in adipose tissue was higher than that of free FAHFAs. They speculate that FAHFA-TAGs act as a "reservoir and buffer" of FAHFAs in organisms, maintaining the stability of FAHFAs content in cells by regulating the balance between fatty acid esterification and TAGs hydrolysis. adipose triglyceride lipase (adipose triglyceride lipase, ATGL) is the "switch" that controls the hydrolysis of FAHFA-TAGs.
The discovery of FAHFA-TAGs has enriched the understanding of the storage form of FAHFAs in organisms. In addition to the FAHFA-TAGs that have been found, are FAHFAs bound in other lipid molecules, such as diglycerides, phospholipids, and cholesterol esters? If so, what is the distribution of this FAHFAs-bound lipid in the organism? What kind of function does it have? These problems remain to be studied.
2.4 FAHFAs analysis method
As previously mentioned, FAHFAs have many important physiological functions in mammals; The study of these functions depends on the efficient FAHFA analysis technology. In the past five years, several methods for targeted absolute quantification and non-targeted full analysis of FAHFAs in complex samples have been developed based on liquid chromatography-mass spectrometry (LC-MS, LC-MS/MS or LC-MS3), which have basically achieved stable, reliable, rapid and highly sensitive absolute quantitative analysis of FAHFAs and screening for FAHFAs.
2.4.1 FAHFAs absolute quantitative analysis
The concentration of FAHFAs in organism is very low (about 0.2 ~ 15 nmol/L in human serum; About 10-2500 pmol/G in mouse adipose tissue) and the matrix composition of biological samples is complex, so it is usually necessary to pretreat biological samples to enrich and purify FAHFAs before detection
[2]. In addition, each family of FAHFAs contains multiple isomers with the same molecular mass and similar chemical properties, so it is a great challenge for the resolution of analytical methods to separate them and achieve simultaneous detection.
In 2016, Kahn et al. Reported an LC-MS method for the quantitative detection of FAHFAs, which is consistent with the analytical method used in the work of Yore et al
[23][2]. The basic process is as follows: firstly, lipid compounds in a biological sample are extracted by using a Bligh-Dyer lipid extraction technology; Then, solid-phase extraction (SPE) was used for further purification. The step is mainly based on the principle of normal phase chromatography, silica gel is used as an extraction column filler, n-hexane and ethyl acetate are used as solvents, most of neutral lipid impurities such as triglyceride, diglyceride and the like in the lipid extract are removed, and the interference to the detection of FAHFAs is reduced; Finally, FAHFAs were detected by high performance liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry (HPLC-ESI-QqQ MS). During the LC-MS analysis, they used isocratic elution to achieve baseline separation of FAHFAs isomers, and the total LC analysis time was about 90 min. The method has good reproducibility, but the sample pretreatment process is complex, the analysis is time-consuming, the organic solvent consumption is large, and the detection sensitivity is poor. In order to improve the analysis efficiency, they improved the above method and used a UPLC system with higher separation efficiency. Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm, Waters) chromatographic column was used to separate FAHFAs. On the basis of maintaining the original separation resolution, the chromatographic analysis time was shortened from 90 min to 30 min (Figure 4)
[24]. The faster analysis speed is conducive to the realization of high-throughput analysis of FAHFAs.
In order to achieve rapid and automated analysis, L López-Basc Bascón et al. Established a method for the detection of FAHFAs in human serum by combining automated solid phase extraction (Auto-SPE) technology with LC-MS analysis
[25]. They used Hysphere C8 cartridges (7 μm, 10 × 2.0 mm, Spark Holland) as SPE extraction columns to construct an online enrichment method to achieve the effect of automatic enrichment and separation analysis at the same time. The application of Auto-SPE makes the operation more rapid and automatic, reduces the pressure of sample pretreatment, and reduces the time of sample analysis (the detection time of each sample is about 25 minutes), but its detection sensitivity is limited, and it can only separate FAHFAs family, but can not separate positional isomers.
Although the above analytical methods can achieve the absolute quantification of FAHFAs in complex samples, the analytical sensitivity is low, which is not conducive to the detection of low-abundance FAHFAs. The reason for the low detection sensitivity of FAHFAs in ESI-MS can be attributed to the carboxylic acid group in the structure of FAHFAs, which is usually detected in negative ion mode and has low ionization efficiency, thus limiting the detection sensitivity. In order to solve this problem, analytical chemists have introduced chemical derivatization and post-column solvent sensitization into the detection of FAHFAs to improve the detection sensitivity of FAHFAs.
Chemical derivatization technology is to label FAHFAs with carboxylic acid derivatization reagents, and to add mass spectrometry sensitizing groups (such as basic groups which are easy to ionize in positive ion mode) to the structure of FAHFAs, so as to improve their ionization efficiency in ESI and enhance mass spectrometry response
[26,27]. Using this technique, our group developed a highly sensitive method for the quantification of FAHFAs
[16]. The overall analysis strategy is shown in Figure 5. In this strategy, we used strong anion-exchange solid-phase extraction (SAX SPE) to separate and purify the extracts of FAHFAs from actual samples. In addition, FAHFAs were labeled with a pair of isotopically labeled reagents, N, N-dimethylethylenediamine (DMED) and d
4-DMED. The tertiary amine group contained on the DMED can be used as a sensitizing group,
And that response of the mas spectrum is improved; The FAHFAs standard labeled with d4-DMED was used as an isotope internal standard to correct the mass spectrum signal. After labeling with DMED, the detection sensitivity of FAHFAs was improved by about two orders of magnitude, and the chromatographic analysis time was also shortened (the basic separation of 16 FAHFAs could be completed within 20 min). The method is suitable for the absolute quantitative analysis of FAHFAs in various biological samples.
Based on the derivatization technology, Han et al. Also proposed a multi-dimensional mass spectrometry shotgun lipoomics (MDMS-SL) method combined with chemical derivatization to detect FAHFAs in biological samples
[28]. N- [4- (aminomethyl) phenyl] pyridine (AMPP) was used as a derivatization reagent for the amidation of FAHFAs, and the quaternary ammonium group in its structure significantly increased the mass spectral signal of FAHFAs (Figure 6). The method is characterized in that the sample is directly subjected to ESI-MS analysis without chromatographic separation after pretreatment and chemical derivatization, so that the analysis time can be greatly shortened; and the method has the disadvantage that only one FAHFAs family can be subjected to overall qualitative and quantitative analysis, and isomer separation and quantification cannot be completed.
In 2019, Cindri Cindrić et al analyzed the contents of 16 FAHFAs in about 20 kinds of foods (apples, oranges, etc.) By using Nano-LC and post-column solvent sensitization combined with ESI-MS technology
[29]. They used ethyl formate as a post-column mobile phase modifier to improve the ionization efficiency of FAHFAs in negative ion mode, thus achieving a detection sensitivity comparable to that in positive ion mode. By analyzing the levels of FAHFAs in these 20 foods, they found that oats, citrus, garlic and pineapple had the highest levels of FAHFAs. SAHSAs are most abundant in foods with antioxidant and anti-inflammatory effects. PAHSAs (previously shown to have hypoglycemic and insulin-sensitive effects) were not detected in certain foods, such as radish, pear, mango, lemon, cherry tomato and kiwi fruit
[2,8].
2.4.2 Screening Analysis of FAHFAs
Previous studies have shown that there are abundant fatty acids and hydroxy fatty acids in organisms, which may provide important precursors for the synthesis of FAHFAs. Fiehn et al. Used computer simulation to predict the structure of FAHFAs that may exist in nature, and the results showed that the number of FAHFAs molecules in nature may exceed 1000
[30]. In order to improve the molecular species information of FAHFAs, two FAHFA non-targeted screening assays have been developed for the discovery of novel FAHFAs molecules.
In 2018, our group developed a strategy of chemical isotope labeling assisted liquid chromatography-mass spectrometry (CIL-LC-MS) to screen and analyze FAHFAs in rice and Arabidopsis
[31]. The overall strategy is shown in Figure 7. After sample pretreatment, rice and Arabidopsis thaliana tissues were labeled with DMED and d
4-DMED reagents respectively. Subsequently, the two labeled samples were mixed 1:1 for LC-MS analysis. In mass spectrometry, we designed possible FAHFAs parent ions and daughter ions according to the fragmentation rules of FAHFAs labeled by DMED and multiple reaction monitoring, and then detected possible FAHFAs in samples by triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. The peaks with the same retention time and similar mass spectrum signal intensity in the MRM scanning mass spectrum of a pair of "light"/ "heavy" labels were recorded, which were potential FAHFAs compounds. Using this method, we detected a total of 49 FAHFA families (262 isomers) in rice and Arabidopsis thaliana, of which 41 FAHFAs families were reported for the first time. This discovery expands the known family of FAHFAs and suggests that FAHFAs are not only a class of mammalian lipid molecules, but also widely present in plants.
Zhang et al. screened FAHFAs in adipose tissue of hamsters using ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS)
[32]. Q-TOF MS can simultaneously acquire primary and secondary high resolution mass spectra of compounds, and then screen potential FAHFAs based on their precise molecular weights and fragmentation characteristics. Ultimately, they detected a total of 17 FAHFAs families (64 isoforms) in hamster fat, of which 9 families were reported for the first time.
The establishment and application of screening methods for FAHFAs have expanded the understanding of the molecular types of FAHFAs. Up to now, researchers have found 75 FAHFAs families in various biological samples, including 11 FAHFAs families found in macrophages and venison by Kuda et al. And Thomas et al., with about 300 isomers (Table 1)
[6,33].
2.4.3 Identification of FAHFAs
Structural identification of FAHFAs is essential to explain their biochemical relevance. The common identification methods of FAHFAs include standard comparison method, metabolic library search method and mass spectrometry analysis method. The standard comparison method is suitable for the identification of FAHFAs with commercially available standards. Mass spectrometry can provide information on the family assignment of FAHFAs, but it is difficult to determine the position of the ester bond of the low-abundance isomers
[34]. In terms of databases, up to now, 103 molecular structures of FAHFAs have been recorded in the Human Metabolome Database (hmdb, https://HMDB.ca/) and Lipid Map Database (http://www.lipidmaps.org/), which provide important reference for the identification of FAHFAs.
In addition to the above commonly used metabolite identification techniques, our research group studied the relationship between the retention behavior of FAHFAs on reversed phase chromatographic column (C18 column) and their structures, and found two chromatographic retention rules of FAHFAs, the ester bond position rule and the carbon number rule
[31]. In the same family, when the ester bond of FAHFAs isomers is closer to the carboxylic acid group, the retention on C18 column is stronger, and the retention factor (log
10k) is linearly related to the position of the ester bond. Rule of carbon number: When FA or HFA on the structure of FAHFAs is fixed, the retention time increases linearly with the increase of the carbon number of FA or HFA. Based on these two rules, we developed predictive models for identifying the structures of positional isomers from different FAHSAs families (Fig. 8). In addition, in order to assist the identification of FAHFAs, our group has recently developed a computer simulation database of DMED-FAHFAs in cooperation with Fiehn et al. The mass spectrum database covers 264 FAHFAs family species, including 4290 high-resolution tandem mass spectrum data
[35].
In terms of absolute quantitative analysis, the developed FAHFAs quantitative analysis methods can basically achieve the detection of FAHFAs in milligram biological samples. In the future research of FAHFAs quantitative analysis methods, simplicity, rapidity, high throughput, better separation resolution and higher detection sensitivity are still the goals pursued by FAHFAs quantitative analysis technology. A good separation of FAHFAs isomers is very important for the study of the specificity of FAHFAs isomers, and the development of more sensitive FAHFAs analysis methods will be conducive to exploring the level of FA HFAs at the cellular level, which is of great significance for the study of the mechanism of action of FA HFAs. In terms of screening and analysis of FAHFAs, it is very important to conduct in-depth screening of FAHFAs in view of their molecular diversity and the fact that different organisms contain different FAHFAs. This is not only helpful to discover new FAHFAs molecules, but also of great significance to improve the understanding of FAHFAs molecular types. The structure identification of FAHFAs is the key and difficult point of FAHFAs analysis. Although the qualitative techniques based on chromatographic retention rules developed by us can solve the problem of identification of partially saturated FAHFAs to a certain extent, it is still very difficult to identify the widely existing unsaturated FAHFAs. Therefore, how to realize the structural identification of FAHFAs composed of unsaturated fatty acids will be the direction of future FAHFAs analysis.
2.5 Chemical synthesis of FAHFAs
The synthesis of FAHFAs is helpful for the structure identification and biological function study of FAHFAs
[36]. At present, there are three main chemical synthesis methods of FAHFA reported.
(1) Yore et al. First reported the synthesis of FAHFAs in 2014
[2]. The basic chemical synthesis steps are shown in scheme 1: (1) tetradecanal is used as a raw material and reacts with 4-pentenylmagnesium bromide to obtain the corresponding alcohol; (2) that alcohol is esterify with hexadecanoic acid anhydride to obtain alkene ester; And (3) finally, carry out ozone oxidation reaction and Pinnick oxidation reaction on that alkene ester to obtain the 5-PAHSA. The advantage of this route is that the reaction steps are less, but the disadvantage is that the yield is low, the atom utilization rate is not high in the esterification reaction, and the price of tetradecanal is expensive.
(2) In 2016, Balas et al. Developed a second method for the synthesis of FAHFAs
[37]. The basic chemical synthesis pathway is shown in Scheme 2. Take the synthesis of 9-PAHPA as an example: ① TBSCl is used to protect the hydroxyl group of enol with TBS; (2) the alkenyl at the end generates an epoxy compound under the action of mCPBA; (3) that epoxy compound is subject to ring-opening reaction under the action of alkynyl lithium and boron trifluoride to obtain beta-alkynol; (4) the alkynyl group of the alkynol is reduced by hydrogenation to obtain the corresponding secondary alcohol; (5) that secondary alcohol is subject to a Steglich esterification reaction to generate ester; (6) remove TBS protection under that action of TBAF to obtain primary alcohol; (7) the primary alcohol generates the corresponding carboxylic acid, namely 9-PAHPA, through the epoxidation reaction. The advantage of this method is that it can be used to synthesize a variety of FAHFA homologues, but the disadvantage is that chromium is used in the last step of synthesis, which has certain chemical toxicity.
(3) In 2017, Nelson et al reported the synthesis of 9 (R) -PAHSA and 9 (S) -PAHSA enantiomers (Scheme 3)
[38]. They used a chiral pool strategy to ensure the desired hydroxyl structure. (1) regioselective ring-opening and alkylation of optically pure epichlorohydrin with a Grignard reagent under the promotion of copper; (2) regenerate epoxide under alkaline condition; (3) another Grignard reagent is used to repeat the above steps for regioselective ring opening to obtain the corresponding alcohol; (4) carrying out esterification substitution reaction and oxidation reaction on the alcohol to finally synthesize 9 (R) -PAHSA. The total yield of the reaction was about 35% and the purity of the product obtained was 98%. Similarly, the 9 (S) -PAHSA enantiomer can be produced using R-epichlorohydrin.
The synthesis of FAHFAs is very important for their analysis and physiological function research. FAHFAs standards are not only the basis of analysis and research, but also an essential tool for the study of physiological functions and metabolic pathways. At present, the developed chemical synthesis methods of FAHFAs can basically meet the needs of general standard synthesis. Future developments in the synthesis of FAHFAs will lead to improvements in the direction of simpler, cheaper and higher yields. In view of the potential of FAHFAs as therapeutic agents for diabetes, it is important to realize the synthesis of FAHFAs in high yield and in large quantities. In addition, besides the chemical synthesis of FAHFAs, biosynthesis is also a promising synthesis technology. The method has the advantages of mild reaction conditions, high reaction yield, little environmental pollution, easy scale-up production and the like. Unfortunately, the research on the catalytic synthetase of FAHFAs is not yet mature, and the research on this technology in the synthesis of FAHFAs still needs further exploration.
3 Conclusion and prospect
With the deepening of FAHFAs research, the influence of FAHFAs in the field of lipidomics research is gradually expanding. At present, the physiological function, metabolism, storage form, analysis and synthesis of FAHFAs have been studied abundantly, and some achievements have been made. In the study of the physiological function of FAHFAs, from the initial study of anti-T2D and anti-inflammatory function to the study of the relationship between FAHFAs and various chronic inflammatory diseases, some interesting phenomena have been observed. Unfortunately, many of the mechanisms behind these phenomena are still unclear, and the metabolic pathways and synthetic enzymes of FAHFAs have not been identified, which is also an important direction for future research on FAHFAs and is crucial for explaining the physiological functions of FAHFAs. In terms of analytical techniques, fast, high-throughput, high-sensitivity and better separation resolution are still the goals of future FAHFAs analysis. Efficient analytical techniques will be of great significance for studying the mechanism of action of FAHFAs at the cellular level and improving the understanding of the molecular types of FAHFAs. Simplicity, cheapness and high yield are the direction of improvement in synthesis technology, which will contribute to the mass production of FAHFAs as drugs in the future.
National Natural Science Foundation of China(21635006)
National Natural Science Foundation of China(31670373)
National Natural Science Foundation of China(21721005)
National Natural Science Foundation of China(21904099)
China Postdoctoral Science Foundation(2018M642893)
PDF
