An Insight to Biological Factors of the Colon Wall: Data Set of Collagen and Elastin Levels Alongside Patient Age, Gender, Colon Segment, and BMI

Introduction:

The colon, a crucial part of the gastrointestinal tract, features a complex structure supported by its extracellular matrix (ECM), primarily composed of collagen and elastin. Understanding the distribution of ECM components like various collagen types and elastin in the colon wall, along with their correlations with demographic factors, is essential for comprehending colon physiology and its implications for gastrointestinal health. These insights can illuminate age-related gastrointestinal disorders, gender-specific conditions, and potential applications in tissue engineering for gastrointestinal reconstruction. The unique aspect of the presented data set lies in its use of samples collected directly from patients during surgical procedures, providing real-world data.

Data Set Overview:

The data set presented here includes information on patients' age, gender, colon segment, and BMI, along with measurements of collagen types I, III, IV, X, and elastin obtained using ELISA. These data are presented in columns 1 to 10 of the dataset. It is important to note that the collagen and elastin values represent the average of duplicate measurements to ensure reliability. Specific inclusion criteria were established here. Patients with common colon conditions such as inflammatory bowel disease (IBD), diverticulitis, or irritable bowel syndrome (IBS) were excluded. The data set includes the data from a study group of 41 patients diagnosed with colon cancer. The initial sample size collected during surgery was primarily determined by the surgeon’s assessment of the colon tumor's characteristics and the necessary margins for safe removal. The surgeon decides on the amount of healthy margin to be excised.

This data set is used to investigate the abundance and interrelationships of various collagen types and elastin in the colon wall, as well as their associations with demographic factors.

In the following, for further clarification of the process of collecting the samples for preparation for ELISA measurement and the process of collagen and elastin measurement using ELISA are presented:

 Collection, Transportation, and Preparation of Tissue Samples

1. Sample Collection: For this study, we collected tissue samples of approximately 10 mm x 10 mm from the colon, ensuring that all samples were taken from regions sufficiently distant from the tumor site to avoid any potential influence of the tumor on tissue characteristics. This approach guaranteed that the samples reflected healthy colon tissue with no tumor-induced pathological changes, allowing for an accurate assessment of collagen and elastin content. Ethical approval was obtained for the study (ethics vote number: 03/20 Universitätsklinikum Magdeburg), and written informed consent was received from all participating patients. During surgery, the colon samples were immediately placed in a 0.9% NaCl solution, stored in an icebox to maintain temperature stability, and carefully transported to the lab, avoiding any direct contact with ice to prevent tissue damage.
2. Sample Storage: Upon arrival at the lab, tissue samples were flash-frozen in liquid nitrogen and stored at -80°C to preserve them for subsequent collagen and elastin level measurements.

Tissue Preparation for Collagen Quantification

1. Thawing and Pre-Processing: Before analysis, the frozen tissue samples were thawed and immediately weighed to record their initial mass. The tissue was then finely minced into pieces ranging from 0.5 to 1 mm using sterile scissors and forceps. Each sample was homogenized in a buffer solution containing 1 mM EDTA, a protease inhibitor cocktail, and 1 mM PMSF. The buffer-to-tissue mass ratio was standardized at 9:1 to ensure consistency across all samples, compensating for variations in initial sample size.

Following homogenization, the lysate was transferred into Lysing Matrix D tubes and processed using the FastPrep-24 5G instrument for 6 cycles, each lasting 40 seconds at 6 m/s. Cooling intervals were applied between cycles to prevent overheating. After disruption, the lysate was centrifuged briefly to separate debris, and the supernatant was transferred to a fresh tube for further analysis. To ensure complete homogenization, the supernatant underwent ultrasonication in three rounds of 10 bursts lasting 1 second each. The lysate was then centrifuged at 6000g for 5 minutes at 4°C to isolate soluble components, which were aliquoted into 4 tubes and stored at -80°C. The pellet was retained for possible further analysis.

Collagen Extraction and Quantification

1. ELISA Assay Preparation: An enzyme-linked immunosorbent assay (ELISA) was used to quantify collagen and elastin content in the samples. A 96-well plate was prepared, with the first 12 wells designated for the standard curve, as each standard was run in duplicate. The remaining wells were allocated to run each sample in duplicate.

Standard Preparation: A stock standard solution was reconstituted with 1 ml of Standard Diluent and allowed to dissolve for 10 minutes at room temperature with gentle mixing to avoid foaming. This reconstituted solution was then diluted to the highest concentration required for the assay. Serial dilutions were prepared by mixing the highest concentration with the Standard Diluent across a series of seven tubes, each containing 0.5 ml of diluent. This process was repeated for each step to produce a range of standards. The final tube, containing only diluent, served as a blank. Each dilution was thoroughly mixed to ensure accuracy. The resulting standards were then used to generate a standard curve, plotting optical density (OD) values against concentration to calibrate the assay.

Standard Curve: OD values were measured at 450 nm and plotted to create a standard curve for each analyte. By matching OD values from the unknown samples against the standard curve, the concentration of the target proteins could be determined. The standard curves were generated for each collagen type and elastin using the corresponding ELISA kits.

ELISA Assay Procedure

1. Assay Execution: All reagents, samples, and standards were prepared in accordance with the provided kit instructions. For each well, 50 µl of either the standard or sample was added, followed by 50 µl of prepared Detection Reagent A. The plate was gently shaken to mix the contents, then incubated for 1 hour at 37°C. After incubation, the wells were aspirated and washed three times with the wash buffer. Next, 100 µl of Detection Reagent B was added, and the plate was incubated again for 30 minutes at 37°C. The wells were aspirated and washed five times before adding 90 µl of Substrate Solution and incubating for 10-20 minutes at 37°C, protected from light. Finally, 50 µl of Stop Solution was added to terminate the reaction, and the OD was immediately measured at 450 nm using a microplate reader.