Elisa kit

Urine strips were inserted into the urine samples up to the test area for less than 2 s. The edge of the strips were drawn along the brims of the vessels to remove excess urine, making sure the tests areas did not touch the vessels. The strips were held vertical and the tips tapped on absorbent papers to remove any remaining urine [14 (link)]. The urine strips were held horizontally and compared with the color charts on the vial label under bright light. The amount of protein was then determined using the intensity of the blue green color which was proportional to concentration of protein in the urine. Proteinuria was defined as the presence of urinary protein with concentration of at least “+” [15 (link)]. The Lipid Profile was determined using the Selectra Pro S (Vital Scientific B.V. Van Rensselaerwweg 4, NL 6956AV Spankeren, The Netherlands) automated chemistry analyzer using the procedure outlined for the equipment while serum zonulin levels were determined in duplicates using human zonulin ELIS AKit (Immundiagnostik AG, Bensheim, Germany).

For urine collection at week 0, 2, and 4, each mouse was housed overnight individually in a metabolic cage with free access to tap water (mouse metabolic cage. NATSUME SEISAKUSHO, Tokyo, Japan). At week 4, the mice were housed in metabolic cages under stabilizing conditions for the collection of 24 h urine samples, and the samples were used for urinary NE as an indirect marker of sympathetic nerve activity using high-performance liquid chromatography (Tosoh Corporation, Tokyo, Japan). Urinary creatinine concentrations were measured by immunoassay (DCA 2000 system. Bayer Diagnostics, Elkhart, Ind., USA). Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) concentration was measured using a highly sensitive ELISA kit (JaICA, Shizuoka, Japan) and then the urinary 8-OHdG/creatinine ratio (μg/g · Cr) was calculated. Urinary angiotensinogen (AGT) concentration was measured using mouse AGT ELISA (code no.27413, IBL, Gumma, Japan), urinary AGT/creatinine ratio (μg/g · Cr) was determined, and the result was expressed as rate of change of urinary AGT. At week 4, plasma levels of ADMA were measured by ELISA kit (Immundiagnostik AG, Bensheim, Germany).

Fecal calprotectin concentrations were measured with a quantitative PhiCal enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, catalog no. K6927) according to the manufacturer’s instructions. Fecal specimens were diluted 1:2,500. ELISA plates were read by the use of a Thermo Scientific microplate reader (Multiskan FC; optical density at 450 nm against 620 nm). Samples containing ≥100 μg of calprotectin per 1 g of feces were considered calprotectin positive (51 (link)).

We analyzed different domains of markers: hematology (e.g., leukocytes), muscle physiology (e.g., bicarbonate, creatinine), metabolic activity (e.g., glucose, insulin) and intestinal function (e.g., zonulin, intestinal fatty acid binding protein). A list of the parameters in the heatmap (Figure 3), including abbreviations, is provided as Supplementary Material (Supplement 2). Hematologic markers were analyzed using Advia 1200 (Siemens). Other markers were measured using the Cobas 6000 (Roche) (Star-shl, Etten-Leur, Netherlands), according to standard procedures. Zonulin concentrations in serum were determined using an ELISA kit (Immundiagnostik AG, Bensheim, Germany) and measured with an ELISA plate reader at 450 nm against 620 nm as reference (Wegh et al., 2019 (link)). Serum intestinal fatty acid binding protein (iFABP) levels were measured using a commercial human ELISA Test Kit (HK406, Hycult Biotech, Uden, Netherlands) according to the manufacturer’s instructions, and analyzed with a multi-detector microplate reader VICTOR^TM X3 (PerkinElmer) using Workout v2.5 software.

The FDA-approved T-Stat (Spectros), was used to measure duodenal mucosal oxygen saturation (D2). Only 47 patient’s mucosal oxygenation could be measured due to technical difficulties (29 hyperglycemic; 18 normoglycemic). After suppressing peristalsis with a single dosage of hyoscine butylbromide (Buscopan- 10 mg/ml), multiple measurements were taken and an average of three readings per individual was used for analysis. The gut permeability assay using serum zonulin was performed using ELISA kit (Immundiagnostik AG, Bensheim, Germany)^{65 (link)}. Only pre-endoscopic sera samples were used to measure zonulin (16 hyperglycemic and 12 normoglycemic). The spearman correlation test was used to test association between zonulin and anthropometric measurements and bacterial load.

The zonulin concentration was determined by using an ELISA kit (Immundiagnostik AG, Bensheim, Germany, batch No. K5601-150513). The assay used the competitive binding technique. Biotinylated zonulin tracer was added to the samples, standards, and controls as a competitor to the sample’s own zonulin. The intensity of the color was inver¬sely proportional to the zonulin concentration in the sample. Samples were read at 450 nm, and the 4-parameter algorithm was used to form the standard curve and to calculate data. All tests were carried out in duplicate. Zonulin concentration is presented in ng/mL. Based on the manufacturer´s studies of serum samples of apparently healthy persons (n = 40), a mean value of 34 ± 14 ng/mL was estimated. Inter-assay coefficient of variance (CV) were 13.3% and 13.6% for the lowest and highest control, respectively, and intra-assay CV were 3.4% and 6.0% for the lowest and highest control, respectively.