Biomarkers in body fluids and their detection techniques for human intestinal permeability assessment
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Jian Zhang
and
Lei Zheng
Abstract
Introduction
Intestinal permeability (IP) is a crucial index for intestinal tract integrity, reflecting intestinal barrier function, injury, diseases, and related systemic conditions. However, current mainstream testing methods are invasive and qualitative. Biomarkers for IP in body fluids offer noninvasive and quantitative advantages for IP assessment while pose challenges for high-performance detection techniques.
Content
This review introduces the main types of biomarkers for IP, including sugar probes, endotoxin, D-lactic acid, intestinal fatty acid-binding protein, diamine oxidase, citrulline, zonulin and lipopolysaccharide-binding protein. Their sources, associated diseases and detection techniques are summarized, and the regulation of intestinal ecosystem and IP by diet are also briefly discussed.
Summary
By investigating existing studies, various biomarkers in body fluids and their detection techniques are introduced, and the effectiveness and realizability of IP assessment with body fluid markers are demonstrated. This review can be as a reference for gastroenterologists, laboratory technicists and researchers.
Outlook
Body fluid biomarker detection provides a promising proach for IP in vitro assessment. To improve the feasibility in clinics, more types of biomarkers, more accurate physiological and pathological mechanisms, and more innovative sensing technologies are expected to be explored and developed. It is foreseeable that fast, precise, and on-site IP assessment will contribute to the clinical diagnosis and monitoring of intestinal and secondary diseases.
Introduction
Human intestine is an important digestive tube from pylorus of stomach to anus, containing small intestine, large intestine and rectum. Its intestinal mucosa consists of multiple layers with specific functions and serves as an interface between the body and the external environment. It is a selectively permeable barrier in direct contact with food, drugs, residues, microbes, antigens, and toxic or carcinogenic substances [1], 2]. The integrity of intestinal barrier is crucial for maintaining the organism health. Many factors such as food, chemicals, gut microbiota, diseases and trauma may cause a disruption of the intestinal barrier [3], [4], [5]. When intestinal contents invade other organs through the damaged intestinal mucosal epithelium, a series of metabolic abnormalities and dysfunctions will occur [6], 7]. Intestinal diseases include inflammatory, necrotizing, functional, and gluten-induced enteropathies [8], 9], which all may affect the intestinal permeability (IP). Secondary organ diseases outside the gut are found in kidney, cerebrovascular, liver [10], 11], etc. For examples, the released lipopolysaccharide (LPS) may activate the immune cells within the liver, produce inflammatory factors and oxidative stress, and promote fat accumulation, inflammation (fatty liver disease) and fibrosis. LPS and inflammatory factors may also lead to insulin resistance related to diabetes. The induced neuroinflammation and misfolded protein transmission can affect the course of neurodegenerative diseases. The metabolites produced by gut bacteria, TMAO (oxidized trimethylamine), will promote the formation of foam cells and increase the risk of thrombosis. Therefore, monitoring and evaluating the IP are of great importance in clinical gastroenterology.
For human intestine deeply located in the abdominal cavity, its clinical sampling and examining are always facing great challenges. Particularly, the small intestine as a free tissue with multiple composite intestinal loops, has anatomical features of long length, curved overlap, and thin wall thickness of only 1–4 mm. As a result, it is often considered a blind spot in gastrointestinal examinations. Although endoscopy is a traditional invasive diagnostic tool to accomplish both observation and sampling, it trends to cause mechanical trauma, leading to complications such as abdominal colic, acute pancreatitis, and even anxiety [12]. Emerging capsule endoscopy can travel and make an observation with the natural peristalsis of intestine, being painless, noninvasive, and radiation-free. However, it is high cost, with large blind area and short retention time resulting in a high misdiagnosis rate [13]. Limited by the superficial examination, deep tissues of the gut cannot be observed using different types of endoscopies. A risk even exists to aggravate the obstruction [14]. Therefore, its large-scale application is hindered. The mainstream noninvasive detection methods include barium meal X-ray scanning, computer tomography scanning, radioactive nuclide scanning and selective abdominal angiography, which are image based and qualitative, with possible contrast agent allergy and radiation damage particularly for pregnant women and children [15], 16]. All the above medical imaging methods are effective only in areas with significant tissue morphology changes, and have significant limitations in the early diagnosis and directly quantitative evaluation of IP.
In recent years, there is growing concern about the non-intrusive, non-imaging, quantitative and easy-to-use techniques for IP assessments. As a result, gut related biomarkers in body fluids have been receiving increasing attention in both research and clinics. Numerous studies have shown that the IP increasing caused by intestinal barrier damage leads to the concentration change of such markers in body fluids. Therefore, these markers can work as indicators for IP related to early lesions in intestinal mucosal tissue [17]. Specific biomarkers in body fluids as well as their determination techniques provide a new strategy to quantify IP for prevention, diagnosis, and monitoring of related gut diseases, and can even work for immunomodulation and drug screening [18]. This review tries to introduce representative IP biomarkers along with their sources, disease relationships and detection methods, to provide references for body fluid based IP assessment and disease diagnosis.
Intestinal barrier and intestinal permeability
Intestinal barrier is the interface for the exchange of substances between internal and external environments of the body, and consists of the mucus layer, the epithelial cell layer, and the lamina propria, as shown in Figure 1. The mucus layer is the first defense line of the intestine to resist microbial contact with the epithelial cell layer. The epithelial cell layer is formed by tightly connected cells of different types [19], 20], preventing the transmembrane microorganism, processing intestinal and extra-intestinal signals, and regulating adaptive immunity in the human body [21]. As for the lamina propria, it has a function of immune defense containing immune cells [22]. Impaired intestinal barrier means that the permeability of the mucus layer is increased, the junctional complexes between epithelial cells are broken, and harmful bacteria, viruses, and other hazardous substance will enter the lamina propria [23]. When a large number of pathogens continuously cross the epithelial layer and reach the lamina propria, they are difficult to be phagocytosed by immune cells in time. Thus the escaped pathogens will spread into the host systemic circulation and induce various diseases [24].
Intestinal tissue and its intestinal permeability.
IP refers to the capability that allows or prevents the passage of some substance between the intestinal cavity and the tissues, and can be used to evaluate the intestinal barrier function. The increase of IP often has a strong correlation with impaired barrier function due to the disrupted tight junction of intestinal epithelial cells and the widened intercellular gap. Substances in the intestinal lumen pass through the epithelial cell layer mainly in two modes of paracellular and transcellular [25]. The paracellular pathway through the tight junction is commonly used to transport hydrophilic substances, such as lactulose and Ca2+, while the transcellular pathway transfers bacterial products or proteins such as endotoxin and fatty acid-binding protein (FABP) through the intestinal epithelial cell layer with the help of transporter proteins and transporter channels [26]. Therefore, IP can be evaluated by detecting the concentration of various biomarkers in body fluids, and even the types of intestinal barrier impairments can be diagnosed in some degree [27].
Biomarkers and their detection methods for IP assessment
Biomarkers which can be employed to assess IP are divided into two categories of exogenous and endogenous substances. Exogenous substances mainly include sugar probes and products of the intestinal flora, such as endotoxin and D-lactic acid, while endogenous substances in the human body mainly include diamine oxidase (DAO), intestinal fatty acid-binding protein (I-FABP), citrulline, zonulin and lipopolysaccharide-binding protein (LBP).
Sugar probes
Sugar probes are generally made of safe and natural sugar substances. They are metabolically inert in the human body, and after intestinal absorption, they are excreted from the urine with original molecules, which can directly reflect IP [28]. The widely adopted sugar probes are lactulose and mannitol (L&M). Lactulose is a disaccharide synthesized from galactose and fructose with a molecular radius about 0.42 nm that can be microabsorbed into the systemic circulation through the small intestine. Mannitol is a monosaccharide with a molecular radius smaller than 0.4 nm that can freely cross the intestinal barrier, and the absorption rate is not affected by the status of the intestinal barrier [29]. After oral administration of L&M, the proportion of L: M in the normal human urine is≤3: 100. When the intestinal barrier is damaged, this proportion will increase [30]. Therefore, IP can be assessed by measuring the ratio of lactulose to mannitol in urine [31]. However, L&M is susceptible to the catabolism by the bacterial flora in colon, and can only be used for permeability assessment of small bowel. Sucralose is not affected by any catabolism in the human body including the colonic flora [32], 33], and can theoretically be as a marker for indicating the whole gut permeability. Clinically, quantification of single sucralose is susceptible by individual differences in intestinal transport, renal function and urine collection. As a result, excretion ratios of multiple sugar probes such as triple sugar (L&M and sucralose) or multi-sugar may provide a more accurate and comprehensive assessment for the IP of small intestinal, colonic, or whole intestinal tract [34].
Common methods for sugar probe detection include high-performance liquid chromatography (HPLC) [35], gas chromatography (GC) [36] and capillary electrophoresis (CE) [30]. GC is less affected by the interferences but requires complex derivatization for the samples. HPLC is widely adopted for the non-derivatization and the advantages on separation efficiency, reproducibility and detection speed. Particularly, its combination with mass spectrometers (MS), refractive index detectors (RID) and pulsed amperometric detectors (PAD) can further improve the detection speed and precision [37], 38]. However, the required equipment is expensive, and specialized technicians are also necessary. CE with solid-phase extraction and ultraviolet source does not require large instrument and sample derivatization. Besides, enzymatic method is occasional used. It is interfered from other sugars in the urine, and has a high limit of detection (LOD) of mg/mL, unsuitable for high-precision testing [39]. Hereinafter, the research on quantitative sugar probe detection during the last 20 years are introduced.
Detection of L&M is quite widely adopted in clinics. Owing to the low cost and easy operation, CE and enzyme assays are employed for a rough detection. In 2006, Paroni et al. used CE for the first time to quantify L&M concentration in urine from the type I diabetic patients. The detection ranges of L&M were 0.01–0.5 mg/mL and 0.05–2.0 mg/mL, respectively. This method was not of high sensitivity, but only required simple instruments such as fused silica capillary tubes, and did not require any derivatization steps [40]. In 2012, Acar et al. improved the above method from Paroni et al. by utilizing a homemade solid-phase extraction column to further reduce the cost of CE detection while maintain the sensitivity. The detection ranges for L&M were 0.02–0.40 mg/mL and 0.30–2.00 mg/mL, and the LODs were 0.0076 mg/mL and 0.14 mg/mL, respectively [30]. In 2014, Linsalata et al. used an enzymatic assay to quantify L&M in urine from the celiac disease (CeD) patients, with the detection ranges of 0.1–16 mM and 0.1–20 mM, and the LODs of 0.03 and 0.04 mM, respectively [39].
As for high-precision detection of L&M, Linsalata et al. in 2014 coupled high-performance anion exchange chromatography with PAD applied in urine samples from CeD patients, with the LODs three orders of magnitude lower than that using enzymatic method, i.e. 0.06 and 0.07 µM, and the detection range was 0.3–80 µM [39]. In 2004, Liu et al. developed a HPLC/RID/MS technique for L&M detection in urine from the spontaneous ascitic fluid infected and sterile ascitic fluid patients. The L&M were determined in the range of 5–1,000 μg/mL, with the LODs of 1.40 μg/mL and 1.65 μg/mL, respectively. The results showed a good correlation between small intestinal permeability and disease course [41]. In 2012, Kubica et al. used HPLC-MS/MS to quantify L&M in urine from the children with digestive tract diseases. With a high sensitivity, the LOD of L&M were 15.94 ng/mL and 11.48 ng/mL, respectively. The detection range was from 50 to 2,000 ng/mL [42]. With the development of MS technology, the resolution, signal-to-noise ratio and scanning rate were significantly improved. In 2022, Sequeira et al. employed a resin-based column to improve the conventional HPLC-RID, increasing the ion separation efficiency and inhibiting the interferences in RID. The detection range of L&M in spiked urine was 0–500 μg/mL, and the LOD was 15 μg/mL [29]. As recently as 2025, Magalhães et al. detected L&M in urine from malnourished children using HPLC-MS/MS method. The LODs of L&M were as low as 5.5 and 0.3 pg/mL, respectively, with a detection range of 10–2,000 ng/mL [43].
In contrast to the well-established assessment of small intestinal permeability via L&M, the assessment of whole intestinal permeability using sucralose or multi-sugar is still within the laboratory exploration. In 2003, Farhadi et al. used capillary column gas chromatography (CCGC) to quantify L&M and sucralose in urine from the healthy volunteers. The respective LODs are 0.5 mg/L, 1 mg/L, and 0.2 mg/L, with the corresponding detection ranges of 0.0005–2 g/L, 0.01–40 g/L, and 0.001–4 g/L. They studied the metabolic rate of mannitol in the human colon, and proposed adopting the ratio of sucralose: mannitol as an indicator for overall IP [44]. In 2005, Anderson et al. spiked sucralose into the urine from 60 randomized hospitalized patients, and tested the samples using HPLC-RID. The detection range was 25–500 mg/L and the LOD was 11 mg/L [33]. In 2006, Farhadi et al. first addressed the interference from other sugars in CCGC. They shortened the detection time (within 22 min) and avoided interferring from common dietary sugars (lactose and fructose) to L&M by reducing the thickness and increasing the length of the capillary column [45]. In 2011, Lenaerts et al. developed a novel method based on isocratic cation exchange LC-MS for triple sugar quantification in urine and plasma from healthy volunteers in the range of 1–1,000 µM, with the LODs of 0.1 µM for mannitol and 0.05 µM for lactulose and sucralose [46]. However, all of the above reports did not perform any clinical validations for IP. In 2022, Sarotra et al. achieved simultaneous detection of sucrose (a marker for gastric permeability broken down after entering the intestine), L&M, and sucralose in urine from the patients with ulcerative colitis using HPLC-RID. The LODs of the four sugars were 78.8 mg/L, 85 mg/L, 74.8 mg/L and 50.9 mg/L, respectively, indicating a feasibility of multi-sugar assay for clinical assessment of whole gastrointestinal permeability [47]. This became the first report for clinical gastrointestinal permeability assessment using four sugars.
In summary, multi-sugar assay is a promising approach for noninvasive assessment of whole intestinal permeability, but most work focus on methodological research. Researchers are developing new detection techniques, investigating their clinical performance, and revealing the relation between multi-sugar probes and intestinal diseases, which will provide a basis for early screen, prognostic assessment, and treatment decisions for intestinal barrier dysfunction. The research on sugar probes, detection techniques as well as their performance during the last 20 years are listed in Table 1.
Comparison of analytical characteristics of different detection methods using disaccharide probes.
| Biomarkers | Detection method | Detection sample | Linear range | LOD | LOQ | Analysis recovery range | Reference |
|---|---|---|---|---|---|---|---|
| Sucralose | HPLC-RID | Spiked urine | 25–500 mg L−1 | 11 mg L−1 | – | 103.1 % | [33] |
| Lactulose/Mannitol | CE | Patient’s urine | 0.02–0.40 mg mL−1/0.30–2.00 mg mL−1 | 0.0076 mg mL−1/0.14 mg mL−1 | 0.02 mg mL−1/0.3 mg mL−1 | – | [30] |
| Colorimetric/enzymatic | Urine of CeD patients | 0.1–16 mM/0.1–20 mM | 0.03 mM/0.04 mM | – | 140–95 %/130–96 % | [39] | |
| HPAC-PAD | Urine of CeD patients | 0.3–80 µM | 0.06 µM/0.07 µM | – | 97–102 % | [39] | |
| GC | Urine of burn patients | 5–50 nmol/50–500 nmol | 1 nM/5 nM | – | 101–112 %/97–106 % | [36] | |
| HPLC-RID | Urine of patients with spontaneous ascites infection | 5–1,000 μg mL−1 | 1.40 μg mL−1/1.65 μg mL−1 | – | 93.1–97 % | [41] | |
| HPLC-MS/MS | Urine of children with digestive tract diseases | 50–2000 ng mL−1 | 15.94 ng mL−1/11.48 ng mL−1 | 47.83 ng mL−1/34.43 ng mL−1 | 95.06–99.92 % | [42] | |
| HPLC-MS/MS | Urine of malnourished children | 10–2000 ng mL−1 | 0.0055 ng mL−1/0.0003 ng mL−1 | 0.0168 ng mL−1/0.0010 ng mL−1 | 95.7–99.8 %/111.6–132.2 % | [43] | |
| Lactulose/mannitol/sucralose | CCGC | Urine of healthy volunteers | 0.0005–2 g L−1/0.01–40 g L−1/0.001–4 g L−1 | 0.5 mg L−1/1 mg L−1/0.2 mg L−1 | – | – | [44] |
| PCGC | Urine of healthy volunteers | 0.025–2 g L−1/0.5–40 g L−1/0.05–4 g L−1 | 25 mg L−1/500 mg L−1/50 mg L−1 | – | – | [44] | |
| LC-MS | Urine and plasma of healthy volunteers | 1–1000 µM | 0.05 µM/0.1 µM/0.05 µM | – | 90–110 % | [46] | |
| HPLC-RID | Urine of patients with ulcerative colitis | – | 84.994 mg L−1/74.789 mg L−1/50.908 mg L−1 | 283.31 mg L−1/249.30 mg L−1/169.69 mg L−1 | 95–130 %/99–146 %/101–129 % | [47] |
Endotoxin
Endotoxin generally refers to the lipopolysaccharide (LPS), which is a major component of the outer cell wall of Gram-negative bacteria. It has a molecular weight greater than 10 kDa and is thermally stable. Numerous Gram-negative bacteria in the human gut release endotoxin into the intestinal lumen. When the intestinal barrier is intact, endotoxin cannot enter the systemic circulation [48], otherwise it will enter the bloodstream. In clinical studies, endotoxin has been served as a typical biomarker for assessment of many diseases, such as steatohepatopathy [49], colon and rectum perforations [50]. Therefore, detection of endotoxin in human blood is of high value for evaluations of IP, enteropathy and parenteral diseases.
Endotoxin can be quantified by measuring its concentration or activity in blood, with a clear relation between its activity and content (1 EU ≈ 0.2 ng). Traditional detection methods include rabbit pyrogen test (RPT) and limulus amoebocyte lysate (LAL) recognized by the U.S. Food and Drug Administration [51]. RPT determines the amount of injected endotoxin by observing the body temperature change of rabbits within a few hours [52]. This test with a LOD of 0.5 EU/mL is indirect and time-consuming, and is mainly used for the safety evaluation of endotoxin in material extracts, not suitable for the detection in body fluids [53]. The LAL tests include gel-clot, turbidimetric, and colorimetric methods [54]. On contact with high-concentration endotoxin, the LAL will coagulate, and the gel-clot method can only make a rough measurement of endotoxin [55]. However, when LAL reacts with a small amount of endotoxin, it will become turbid, and its turbidity can be directly proportional to the endotoxin content. Therefore the endotoxin in fluids can be estimated by the standard turbidity curve. For colorimetric method, it is based on the activation of LAL clotting enzyme with endotoxin to cleave certain synthetic amino acids and release p-nitroaniline. The maximum absorbance of the reaction, proportional to the endotoxin concentration, is measured at the wavelength of 405 nm using a microplate reader [56]. Turbidimetric and colorimetric methods both have low LODs at 0.001 EU/mL level [57], and the detection time is usually within 2 h [58]. Rojo et al. and Gardiner et al. measured endotoxin using LAL and verified that the course of inflammatory bowel disease (IBD) and the probability of its complications (systemic endotoxemia) are significantly related to the increase of IP [59], 60]. However, LAL assay is prone to interference from the heterotoxin as well as the cell wall products from fungi, Gram-positive bacteria, and so on. As a result, the indicative value of endotoxin in clinical setting may not be sufficient [61]. Nevertheless, techniques such as immunology, fluorescence and emerging biosensors have been utilized to improve the sensitivity and accuracy of endotoxin detection, as described below.
In 1998, Romachin et al. developed a chemiluminescence assay for endotoxin based on the antigen-antibody complex and subsequent complement-mediated neutrophil reaction in blood. This method had a detection time of shorter than 30 min, a detection range of 0.017–1.6 EU/mL, and a LOD of 0.017 EU/mL [62]. Chemiluminescent analysis was also used in clinics. In 2021, Matsuda et al. performed the endotoxin detection in blood from the patients with colorectal perforation, and verified the feasibility of endotoxin detection in blood for enteropathy diagnosis [49]. In 2009, Mitsumoto et al. utilized a laser light-scattering platelet aggregometer to detect light-reflecting particles produced by the reaction between LAL reagent and endotoxin. This method was more resistant to interference and more sensitive than turbidimetry, with a LOD of 0.00015 EU/mL and a detection time within 71 min [63]. In 2010, Noda et al. reported a method for detecting endotoxin using LAL reaction combined with mutated firefly luciferase. This method had a better signal-to-noise ratio and a higher luminescence intensity than traditional LAL, and could detect 0.0005 EU/mL endotoxin within 15 min [64]. In 2011, Granlert et al. developed an endotoxin-sensitive reagent that can selectively capture endotoxin using phage-derived receptor proteins, with a detection range from 0.05 to 500 EU/mL and a LOD of 0.05 EU/mL. This method improved the specificity affected by the products from the cell walls of fungi and Gram-positive bacteria [65].
As for biosensors, Limbut et al. in 2007 immobilized endotoxin-neutralizing proteins on a gold electrode for determining endotoxin levels in E. coli cultured broth. Compared with the LAL method, the detection time was shortened to 13–18 min, with a LOD of 1.0 × 10−13 M and a detection range of 1.0 × 10−13–1.0 × 10−10M [66]. In 2019, Yu et al. integrated peptide-assembled graphene oxide and DNA-modified gold nanoparticles on an electrochemical sensor for endotoxin in spiked human serum, with a detection range of 0.005–1 EU/mL and a LOD of 0.001 EU/mL [67]. In 2021, Çimen et al. prepared a surface plasmon resonance sensor based on molecular imprinting for real-time detection of endotoxin in spiked artificial plasma. The detection results were close to that from LAL, and the response time was shortened to about 14 min. The detection range was 0.5–100 ng/mL, and the LOD was 0.023 ng/mL [48]. Although these techniques were not yet applied in clinical practice, they exhibited significant advantages on convenience, speed, sensitivity and specificity.
To sum up, endotoxin detection has shown prospect for IP assessment and related disease diagnosis. It is also encouraging that many novel biosensors are being developed, which have obvious merits for quick and easy detection. As a matter of fact, the related studies are mostly of exploratory stage in laboratory, and are limited to the spiked samples, but the novel techniques with powerful performance are still emerging, and further clinical IP assessment and enteropathy diagnosis might be one step away. The typical reports on endotoxin detection are listed in Table 2.
Comparison of parameters for different endotoxin detection methods.
| Method | Detection sample | Linear range | LOD | Detection time | Reference |
|---|---|---|---|---|---|
| Whole blood assay | Whole blood samples | 0.017–1.6 EU/mL | 0.017 EU/mL | 30 min | [62] |
| Laser light-scattering particle-counting | Standard sample | 0.0005–0.05 EU/mL | 0.00015 EU/mL | 71 min | [63] |
| Biolumines-cence test | Standard sample | 0.0005–0.1 EU/mL | 0.0005 EU/mL | 15 min | [64] |
| EndoLISA | Standard sample | 0.05–500 EU/mL | 0.05 EU/mL | – | [65] |
| Capacitive biosensor | E. coli culture solution | 1.0 × 10−13 to 13−1.0 × 10−10 M | 1.0 × 10−13 M | 13–18 min | [66] |
| Electrochemical biosensor | Serum | 0.005–1 EU/mL | 0.001 EU/mL | – | [67] |
| Surface plasmon resonance sensor | Artificial plasma | 0.5–100 ng/mL | 0.023 ng/mL | 14 min | [48] |
D-lactic acid
D-lactic acid is mainly caused by lactic acid-producing bacteria in the intestines and is present only in small amounts in body fluids of healthy individuals [68]. Intestinal flora imbalance causes an proliferation of various acid-producing bacteria, making an increased D-lactic acid after the fermentation of carbohydrates in the intestines. Excessive D-lactic acid will enter the bloodstream through the damaged intestinal mucosa, and can trigger systemic reactive inflammation. Commonly, serum D-lactic acid is considered as a typical indicator for mesenteric ischemia and intestinal infarction.
Enzymatic assays are the common methods for determining D-lactic acid in body fluids, with the reaction product of reduced nicotinamide adenine dinucleotide (NADH), which is proportional to the D-lactic acid concentration, and can be quantified by the absorbancy using spectrophotometry [69]. IP increase caused by IBD [70], necrotizing small bowel colitis [71] and acute mesenteric ischemia (AMI) [72] are with significantly higher concentrations of D-lactic acid in blood, demonstrating the feasibility of D-lactic acid evaluation for IP assessment and gastrointestinal disease diagnosis [73].
The sensitivity of traditional enzymatic methods for D-lactic acid is relatively low, with the LODs generally at mM level. Besides, low specificity, long detection time and weak robustness against the environment are accompanying. Therefore, researchers tried to develop novel techniques to improve the performance. In 1992, McLellan et al. replaced the spectrophotometry with fluorescence in enzymatic method to determine the D-lactic acid level in blood samples from the diabetic patients, achieving a lower LOD of 3.73 μM, with a detection range of 0–100 μM [74]. Since NADH is produced not only from D-lactic acid but also from endogenous L-lactic acid, the removal of L-lactic acid is always necessary during the enzymatic assays. In 2005, Tan et al. used CE to determine D-lactic acid in plasma for assessment of increased IP caused by severe trauma. This method improved the specificity but had poor sensitivity, with a detection range of 0.025–5 mM and a LOD of 20 μM [68]. In 2011, Henry et al. used HPLC-MS/MS for D-lactic acid determination in urine with a detection range of 0.5–100 μM. The LOD and limit of quantitation (LOQ) were 0.125 and 0.5 μM, respectively. This method enabled the D-lactic acid quantification at lower concentrations [75]. In 2021, Rasmussen et al. combined an automated analyzer with a D-lactic acid kit, removing the interference and enabling the quantification in plasma within 9 min. It could be used for rapid diagnosis in acute clinical settings with a detection range of 0.05–9 mM and a LOD of 40 μM [76]. Park et al. in 2021 reported a miniaturized 4 × 4 photoelectric sensor array for on-chip quantification of D-lactic acid. Based on the photoconductance change of the molybdenum disulfide channel, this sensor array realized a high-throughput detection and improved the detection efficiency. The detection time was around 10 min, the detection range was 11.1–11.1 mM, and the average LOD in saliva, urine, and serum was 9.7 ± 0.36 nM [77].
Although the emerging sensors have provided powerful approaches for D-lactate test, few clinical trials have reported due to the enzyme activity being easily affected by the environment (temperature, humidity, etc.). This problem might be resolved in the future by strictly controlling the enzyme reaction conditions or adopting other bioprobes, such as aptamers.
I-FABP
I-FABP is a small molecular (12–15 kDa) cytoplasmic protein mainly responsible for promoting fatty acid transport and metabolism in enterocytes. It is specifically expressed in mature epithelial cells of the small intestine, accounting for approximately 2 % of the cytoplasmic proteins in the intestinal epithelium [78]. According to the recent reports, I-FABP is related to non-intestinal diseases, such as metabolic dysfunction-associated steatotic liver disease, chronic kidney disease, and even schizophrenia [79], 80]. Excluding these non-intestinal barrier issues, I-FABP will release and enter the systemic circulation after an injury to the intestinal tissue, and therefore its concentration in systemic circulation will increase [81]. Generally, I-FABP has been recognized as a sensitive indicator for IP and diseases associated with intestinal mucosal damages, such as mechanical small bowel obstruction [82], necrotizing small bowel colitis [83], and ischemic bowel disease [84].
Clinical studies for I-FABP detection mainly occurred after 2010, with the most common method of ELISA. In 2011, Kanda et al. demonstrated that I-FABP level in serum became significantly higher from the patients with ischemic bowel disease. They found that the sensitivity and the predictive value of I-FABP were superior to other markers, contributing to the early diagnosis of intestinal ischemia [85]. In 2013, Güzel et al. investigated the correlation between AMI and I-FABP, showing that I-FABP had a better specificity than the indicators of D-dimer and white blood cell [86]. In 2017, Voth et al. found that I-FABP level in plasma from the patients with intestinal injuries was obviously higher than that from healthy individuals, confirming that I-FABP was sensitive for intestinal injury diagnosis [87]. In 2022, Tyszko et al. verified the plasma I-FABP for reflecting the intestinal injury caused by severe COVID-19 [81]. Non-disease (such as drugs and strenuous exercise) caused intestinal tissue damage and IP increase can also lead to an increase of I-FABP in body fluids [88], 89].
As a matter of fact, ELISA is time-consuming usually with 2–4 h to finish an assay, which cannot meet the bedside rapid testing, particularly for critical patients. Unfortunately, new techniques for I-FABP detection were rarely reported. One typical work was presented by Abdelrasoul et al. in 2018, in which an electrochemical biosensor was prepared to detect I-FABP in urine for noninvasive clinical diagnosis of AMI [90]. Gold nanoparticles was utilized to enhance the detection signal, and the response time was reduced to less than 2 h. Although this electrochemical immunosensor was not so competitive among the electrochemical devices, this sensor still showed a performance improvement for I-FABP detection. Referring to similar detection techniques for other biomarkers, biosensors should have advantages for fast on-site detection of I-FABP.
In brief, I-FABP has been recognized as a good biomarker for assessment of IP and intestinal dysfunction. The I-FABP concentration increasing in blood can effectively indicate the pathologic statuses of the intestinal tracts. However, current clinical pathological research is relatively insufficient, leading to the lack of strict correlation between I-FABP concentration and enteropathies. Meanwhile, rapid and accurate I-FABP detection techniques are uncommon, posing a challenge to large-scale clinical trials. Based on the definite studies of this biomarker, we believe that more attention will be attracted in both pathology research and detection technology development.
DAO
DAO is an intestinal cytoplasmic enzyme specifically expressed at the tip of mature villous cells of the small intestinal mucosa, exhibiting high activity [91]. DAO activity is with a significant correlation to the integrity of small intestinal mucosa. Therefore, it has been considered as a biomarker for assessing small intestinal permeability and can provide valuable information for clinical diagnosis of small intestinal diseases [92], 93].
The early researches on DAO activity detection appeared in the 1950s and 1960s. In 1961, Okuyama et al. extracted delta-1-pyrroline produced by the reaction between DAO and cadaverine-C14. The DAO activity was then determined by tracking the radioactive delta-1-pyrroline using liquid scintillation spectrometry [94]. This radiometric method combined with heparin method was widely used before the 1990s, and was known for directly reflecting the integrity of the small intestine mucosa [95]. However, analyte extraction for this radiometric method is complex and is susceptible to the interference such as ascorbic acid, leading to a poor specificity. In 1992, Kazmierczak et al. assessed the DAO activity in neonatal cord serum by measuring the cleavage rate of the histamine substrate by DAO using a spectrophotometric method. This method avoided the extraction steps, with an upper limit of 200 U/L and a LOD of 2.9 U/L [96]. However, the results from this assay might not be so reliable without consideration of possible interference from ascorbic acid. In 1994, Takagi et al. eliminated the interference by adding ascorbate oxidase. The DAO activity in human serum was then indirectly assessed by detecting the solution absorbancy with a product of methylene blue using colorimetric assay, with an upper limit of 50 U/L and a LOD of 2.8 U/L [97]. Whereafter, this method was adopted as a clinical laboratory tool for investigating the integrity and permeability of intestinal mucosa. For examples, Tsujikawa et al. used this method in 1999 finding that DAO activity in serum from hematological cancer patients decreased with the increase of chemotherapy treatment number, concluding that DAO could act as an effective indicator for the intestinal mucosal damage level of cancer patients [98]. In 2012, Namikawa et al. employed this method to find that DAO activity in plasma decreased after oral administration of anticancer drugs in gastric cancer patients, which was related to the pathological changes of intestinal mucosa caused by anticancer drugs [93]. As for the sensors, Wu et al., in 2017, developed a colorimetric biosensor for rapid detection of DAO, which reduced the detection time to 30 min and the response was linear in the range of 0.15–4.5 mU/mL, with a LOD as low as 0.062 mU/mL [99]. Although these reported methods were applied to clinical assessment, they all indirectly quantified the DAO by activity measurement. Techniques is still lacking at present for directly measuring the actual amount of DAO.
In 2016, Boehm et al. developed a sandwich-type ELISA using a monoclonal antibody and a polyclonal antibody. This strategy quantified the DAO concentration in human blood with a detection range of 0.5–450 ng/mL and a LOD of 0.48 ng/mL [100]. In 2019, Cai et al. used ELISA to find that the DAO level in serum was significantly higher with active Crohn’s disease (CD) than those during the remission, with similar diagnostic efficacy to the other two diagnostic markers for CD, i.e., erythrocyte sedimentation and ultrasensitive reactive protein. This work verified the feasibility of DAO for indicating the progression of CD disease [92].
As a summary, DAO has been demonstrated as an effective biomarker for gut health, and the most common clinical means for DAO detection at this stage is colorimetric method, which indirectly reflects DAO content by determining its activity. The direct quantitative technique for DAO is mainly ELISA, while there is still a great potential to improve the detection accuracy as ELISA is often regarded as a semi-quantitative technique. Meanwhile, rapid and easy-to-operate detection for on-site applications are also needed. To develop new assays and sensors for fast precise quantification of DAO is expected to develop the in vitro IP assessment and intestinal disease diagnosis.
Citrulline
Citrulline is not an essential amino acid for the human body. It is an amino acid being not encoded in DNA, and is only synthesized by glutamine in human intestinal epithelial cells [101]. Normally, the citrulline concentration in human plasma is 30–50 μM [102]. When the intestinal mucosa is damaged causing a decrease of the activity of the intestinal epithelial cells, the citrulline concentration in blood will subsequently decrease [103]. The close correlation between citrulline level in blood and enterocyte status can provide an effective way for IP assessment and enteropathy diagnosis [104].
Based on the reaction between citrulline and diacetylmonoxime, colorimetric methods were commonly used in the early years to complete the citrulline determination [105]. For example, Knipp et al. performed a high-throughput parallel colorimetric assay on a 96-well microtitre plate for standard citrulline samples in 2000, with an LOD of 0.2 nM [106]. However, there are more than 30 amino acids in the body fluids, while conventional colorimetric methods lack reliable target separation techniques. Chromatography can solve this problem and has been widely used in many studies of clinical qualitative diagnosis. Based on chromatography, Lutgens et al. in 2004 confirmed the close correlation between serum citrulline and intestinal damage as well as enterotoxicity in cancer patients, finding that citrulline had a higher sensitivity and specificity for small intestinal epithelial damage than sugar probes [107]. In 2005, Gong et al. found that the serum citrulline in patients with short bowel syndrome was much lower than that in healthy individuals. They revealed the negative correlation between citrulline and surface area and length of the small intestine, suggesting citrulline to be an indicator for reflecting the course of intestinal failure [108]. In 2009, Crenn et al. found that citrulline was a reliable biomarker for intestinal epithelial cell assessment in HIV patients, and its concentration could guide the nutritional therapy [109]. In 2014, they further verified that citrulline could be both pre- and pro-gnostic indicators for acute intestinal dysfunction and sepsis [110].
The development of multiple techniques based on chromatography continuously drove the citrulline detection. In 2008, Rougé et al. determined the citrulline concentrations using GC-MS in the matrices of plasma, erythrocytes and urine, with the detection ranges of 0–200 μM, 0–50 μM, and 0–50 μM, respectively [111]. In the same year, Wu et al. performed the citrulline detection based on HPLC coupled with a fluorescence detector. The body fluids included plasma, breast milk, urine, amniotic fluid, allantoic fluid and so on. This experiment had a detection range of 1–100 μM, with a LOD of 5 nM (in the standard) [112]. Also in 2008, Demacker et al. used ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to quantify the citrulline in plasma, with a reduced test time of 30 min and a detection range of 0.6–2,000 μM [113]. In 2010, Mao et al. quantified citrulline in plasma from the patients with gastrointestinal cancer. Using reversed-phase HPLC-UV, the detection range was 0–1 mM and the LOD was 0.0201 μM [114].
In addition to chromatography, Bicker et al. designed a chemical probe named rhodamine-phenylglyoxal in 2012, which specifically bond citrulline under acidic conditions. The amount of citrulline in serum was assessed by fluorescent protein images, with a low LOD of 0.67 pM [115]. In 2014, Forteschi et al. established a single CE for determination of human plasma citrulline, avoiding expensive and complicated derivatization and improving the sensitivity of conventional electrophoresis. The range of this detection was 10–80 μM, and the LOD was 5 μM [116].
For citrulline, its level in various body fluids is closely related to the small intestinal permeability, and chromatography as well as its improved methods has been validated and applied in clinical diagnosis. Researchers are also developing new methods to overcome the shortcoming of chromatography and to further optimize the detection procedures. However, these new methods are still in the experimental stage with standard samples or spiked samples in body fluids, being not clinically applied on a large scale.
Zonulin
Zonulin is produced in human intestine and liver cells, with a molecular weight of 47 kDa [117], and has the functions of regulating the structural tightness between intestinal epithelial cells, and regulating the mucosal immune response [118]. Gluten substances and harmful intestinal flora trigger the release of excess zonulin from in- testinal cells, causing anomaly of the intestinal epithelial cell gaps, and leading to an increased IP and subsequent immune derangement [119].
After zonulin was discovered by Fasano in 2000 [120], it has been more and more used for IP assessment and course indication for related diseases. In 2019, Demir et al. found a higher level of plasma zonulin from the patients with gestational diabetes mellitus than that from the controls, confirming increased IP due to insulin resistance and inflammation [121]. In the same year, Caviglia et al. validated the high sensitivity of zonulin for assessment of IP in patients with IBD [122]. In 2022, Ghanadi et al. proposed that zonulin could be as an effective guide for the development of various types of liver diseases, which were caused by the immune activation and inflammatory response due to the increased IP and the consequent entry of pathogens and inflammatory factors into the systemic circulation [123]. In 2023, Kim et al. found the higher zonulin levels in serum from the patients with severe asthma induced by intestinal barrier defects than that in controls, demonstrating that zonulin could be used as a biomarker of respiratory diseases through intestinal barrier channel [124].
Clinical quantification of zonulin is currently realized by ELISA. However, researchers have found in their experiments that the commercial ELISA kits for zonulin detection may target multiple proteins and do not have a good specificity [125]. Meanwhile, a variety of non-intestinal diseases can have an impact on the zonulin concentration in body fluids. Therefore, pathological mechanisms still need further investigation to determine the correlation between zonulin and intestinal permeability.
LBP
LBP is an acute-phase response protein secreted by liver cells, adipose tissue, and intestinal cells, playing a crucial role in immune defense and inflammatory responses [126]. Under normal physiological conditions, LBP is stably present in serum at a concentration of 5–15 mg/mL. During infections, traumas, or when the intestinal barrier is impaired, its serum level can rapidly increase by 10–50 times, becoming a sensitive marker for both IP and endotoxemia and systemic inflammatory responses [127].
In 2021, Seethaler et al. conducted a study on the healthy cohort and the obesity cohort [128]. They evaluated the association between potential biomarkers and the L: M value. The results showed that the plasma LBP level was significantly correlated with the L: M. Compared with zonulin, this association was independent of age, body mass index and gender, and there was little variation among individuals. In 2022, Iordache et al. discovered that serum LBP concentration was significantly correlated with the depression score (PHQ-9) of IBD patients, suggesting that the systemic inflammatory response caused by increased IP could affect the central nervous system, potentially serving as a pathological mechanism for depressive symptoms. In the same year, Parenti et al. found that, compared with patients with normal weight and well-controlled asthma, the level of blood LBP in obese asthma patients was significantly higher, suggesting that increased IP may lead to the exacerbation of inflammatory response in asthma besides the verified IP related obesity [129].
Based on the protein characteristics of LBP, the main method for detecting its concentration is still ELISA at present. Although the novel assay methods are hardly reported, the improvement in detection performance of LBP using new sensing techniques is something to be expected. Furthermore, big data and artificial intelligence is of importance to establish disease prediction models between IP and secondary disease based on LBP.
Combined detection of the biomarkers
The detection result of a single biomarker is susceptible to various factors, making it difficult to comprehensively and accurately reflect the intestinal barrier injury. For examples, the D-lactic acid level may be affected by food intake and excessive intestinal bacteria [130], The DAO activity may change due to pregnancy, heparin infusion, malignant tumors or certain chemotherapy drugs [131], and the endotoxin level can also be affected by non-intestinal-related factors [132]. Therefore, the combined detection of multiple serum markers can improve the accuracy of the evaluation of IP.
In 2019, Kong et al. compared the dynamic changes of serum α-glutathione S-transferase (α-GST), DAO, D-lactic acid, citrulline and I-FABP in patients with major abdominal surgeries before and after the operations. The sensitivities of D-lactic acid, citrulline and I-FABP tests were all good, but their specificities were low. The sensitivity of DAO test was not good, while the specificity was extremely high. Meanwhile, the accuracies of D-lactic acid and I-FABP tests were excellent. This significantly improved the diagnostic efficacy of intestinal mucosal barrier dysfunction. This investigation concluded that the combined detection of D-lactic acid, citrulline and I-FABP is significantly more reliable than using a single marker in IP assessment after abdominal surgery. In 2023, Yang et al. adopted the combined serum levels of D-lactic acid, DAO and endotoxin in cancer patients for predicting intestinal barrier damage and intestinal-source infections [133]. The combined levels increased with the ileus degree, and the predictive ability using three markers was significantly enhanced compared with sigle-marker strategies.
In theory and limited practice, the combined detection of multiple serum biomarkers shows advantages for accurate evaluation of IP, as cross validation can counteract many interferences. However in practice, the complex mechanism of intestinal barrier injury often leads to asynchronized changes of different markers, causing contradictory judgments. At the same time, the lacks of standardized multiple thresholds for dynamic monitoring, multi-marker detection platform, and optimization of weight distribution for clinical interpretation are posing a great challenge for further development of combined biomarker detection for IP assessment. Establishing standardized processes, conducting large-scale experiment and constructing new assay platforms will accelerate the clinical multi-marker detection for IP.
Conclusions
Intestinal tract is the most important digestive organ in the body. Intestinal injury and disease will significantly affect the IP, which has close relations to many systemic diseases. Therefore, IP becomes an intestinal barrier function being highly concerned in clinical practice. However, IP is always difficult to characterize because the intestinal tract is hidden within the human body and is not easily to observe. Due to the limitation of conventional examinations, researchers have been seeking various biomarkers in body fluids to reliably indicate the IP level over the years. Meanwhile, novel sensors and assays, as a key to improve the detection performance and effect, play a critical role for efficient and simple in vitro assessment of IP. This review introduces the reported biomarkers, their sources, their relevance to IP and related diseases, and their detection methods.
Based on this review, future research directions are proposed as follows. (1) Utilizing multi-omics technologies, more specific biomarkers for IP and intestinal diseases can be screened, and multi-dimensional IP assessment models can be constructed and validated. (2) The biomarker profiles for disease courses can be built to achieve early warning and precise classification for different diseases. (3) Integrated detection platforms based on emerging techniques such as microfluidics and biochips can be developed to achieve simultaneous detection of multiple markers, with high sensitivity and specificity, and short turnaround time.
Dietary nutrition is an important non-drug intervention strategy for regulating IP. A healthy diet excluding high fat, high carbohydrate, emulsifiers, artificial sweeteners and excessive alcohol helps to establish a balanced microbial population and prevent dysbiosis, extremely effective to keep a normal IP. The Mediterranean diet, characterized by high levels of dietary fiber, polyphenols, unsaturated fatty acids and low-processed foods, has been proven to be an effective dietary pattern for maintaining intestinal health. Future research can further clarify the dose-effect relationships of specific polyphenols, dietary fiber types, etc., and explore their interaction mechanisms with intestinal microbiota metabolites such as trimethylamine-N-oxide and indolepropionic acid, in order to provide scientific basis for improving intestinal health through personalized nutritional plans.
In summary, this review mainly introduces various biomarkers present in human body fluids used for IP assessment, with their sources, the correlations with IP and related diseases, as well as the detection methods. Because many IP biomarkers have not been widely used clinically, the content presented in this review is perspective in some degree. However, we believe that in vitro assessment of IP and diagnosis of intestinal diseases, particularly with novel techniques, has great prospects for the expected noninvasive, quantitative, low-cost, portable and fast-responding merits. It is hoped that this overview can provide an outlook in relative fields.
Funding source: Open Research Fund of State Key Laboratory of Digital Medical Engineering
Award Identifier / Grant number: 2024-M04
Funding source: Zhejiang Provincial Natural Science Foundation of China
Award Identifier / Grant number: Q24F040004
Funding source: Research Project of Zhejiang Provincial Department of Education
Award Identifier / Grant number: Y202351740
Funding source: Wenzhou Basic Scientific Research Projects
Award Identifier / Grant number: G20240049
Funding source: Wenzhou Basic Scientific Research Project
Award Identifier / Grant number: G20240024
Funding source: National Natural Science Foundation of China
Award Identifier / Grant number: 62074047
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Research ethics: Not applicable, as the article is a review.
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Informed consent: Not applicable.
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Author contributions: J. Zhang, T. Wang and H. Qi were the major contributors to the writing of this article. L. Zheng supervised the work and provided critical reviews. All the authors read and approved the final manuscript.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: This work was supported by the Natural Science Foundation of China (62074047), the Open Research Fund of State Key Laboratory of Digital Medical Engineering (2024-M04), Zhejiang Provincial Natural Science Foundation of China (Q24F040004), Research Project of Zhejiang Provincial Department of Education (Y202351740), and Wenzhou Basic Scientific Research Projects (G20240024, G20240049).
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Data availability: Not applicable.
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