Table 2.
Methods for assessment of intestinal permeability, epithelial integrity, and mucus properties
| Method | Test Molecules | Applicable Sites | Biological Sample | Comments |
|---|---|---|---|---|
| Methods of assessing intestinal permeability | ||||
| Measurement of short-circuit current in Ussing chambers | Ion transport | Whole intestine | Biopsies | Invasive, fresh tissue or biopsy material needed, duration of experiment limited to 2 h |
| Dual sugar quantification using mass spectrometry | Oligosaccharides of different MW (e.g., lactulose/ mannitol) | Small intestine | Urine | Time consuming, affected by GI motility, renal function |
| Quantification using mass spectrometry | PEGs, 4,000/400 kDa | Whole intestine | Urine | Equivalent performance to dual sugar test reported, time-consuming |
| 51Cr-EDTA radioisotope activity | 51Cr-EDTA | Whole intestine | Urine | Radioactivity |
| LAL assay | Endotoxin (LPS) | Whole intestine | Plasma | Standardization difficult in human samples |
| Methods of assessing epithelial integrity and intestinal inflammation | ||||
| Mass spectrometry | Citrulline, an epithelial amino acid not incorporated into protein | Small intestine | Plasma | Validated as a useful indicator of loss of small bowel epithelial cell mass in transplant recipients and chemotherapy; not likely to be sensitive enough for healthy subjects |
| ELISA | I-FABP | Jejunum | Plasma | Studies mostly in patients with small intestinal inflammation |
| ELISA | I-BABP | Ileum | Plasma | Studies mostly in patients with small intestinal inflammation |
| ELISA | L-FABP | Whole intestine | Plasma | Expressed in kidney and liver |
| ELISA | Zonulin, claudin 3 (potentially other tight junction proteins) | Whole intestine | Plasma | Few studies |
| Confocal fluorescence microscopy of TJ proteins | TJ proteins | Whole intestine | Biopsy or surgical tissue | Requires confocal microscopy and specialized image analysis methods; labor intensive |
| ELISA | Calprotectin | Whole intestine | Feces | Released by activated neutrophils at inflamed sites; evaluated in colitis studies |
| ELISA | LCN-2 | Whole intestine | Feces | Expression of LCN-2 upregulated in epithelial cells by inflammation; also expressed in neutrophils |
| Quantification by real-time PCR | miRNAs upregulated in inflamed enterocytes | Whole intestine | Feces or plasma | Potential new markers but few studies and mainly in cancer patients |
| Morphological studies using paraffin fixed tissue and H&E staining | Tissue appearance and morphology | Whole intestine | Biopsy or surgical tissue | Invasive, used to assess severity of mucosal damage in patients |
| Methods of assessing mucus thickness and penetrability | ||||
| Fluorescent microscopy of mounted tissue ex vivo | Permeability of fluorescent beads through mucus | Whole intestine | Biopsy or surgical tissue | Fresh tissue required, specialized microscopy set up required |
| Carnoy fixation and mucus detection using PAS/Alcian blue or antibodies | Secreted mucus, | Whole intestine | Tissue sample | Invasive but can be used to measure mucus thickness and quantify goblet cell numbers, can be combined with FISH staining of microorganisms; human biopsy sampling method may not preserve mucus layer |
51
Cr-EDTA, chromium-labeled EDTA; FABP, fatty acid binding protein; FISH, fluorescent in situ hybridization; GI, gastrointestinal; H&E, hematoxylin and eosin; I-BABP, ileal bile acid-binding protein; I-FABP, intestinal fatty acid-binding protein; LAL, limulus amebocyte lysate assay; LCN-2, lipocalin-2; L-FABP, liver-type fatty acid-binding protein; LPS, lipopolysaccharide; miRNA, microRNA; MW, molecular weight; PAS, period acid Schiff; PEG, polyethylene glycol; TJ, tight junction.