Importance of the Evaluation of N-Acetyltransferase Enzyme Activity Prior to 5-Aminosalicylic Acid Medication for Ulcerative Colitis

Abstract

Background: 5-aminosalicylic acid (5-ASA) is a classic anti-inflammatory drug for the treatment of ulcerative colitis. N-acetyltransferase (NAT) enzymes convert 5-ASA to its metabolite N-acetyl-5-ASA, and it is unresolved whether 5-ASA or N-acetyl-5-ASA is the effective therapeutic molecule. We previously demonstrated that colonic production of N-acetyl-5-ASA (NAT activity) is decreased in dextran sulfate sodium-induced colitis. Our hypothesis is that 5-ASA is the therapeutic molecule to improve colitis, with the corollary that altered NAT activity affects drug efficacy. Since varying clinical effectiveness of 5-ASA has been reported, we also ask if NAT activity varies with inflammation in pediatric or adult patients.

Methods: Acute colonic inflammation was induced in C57BL/6 NAT wild-type (WT) or knockout mice, using 3.5% dextran sulfate sodium (w/v) concurrent with 5-ASA treatment. Adult and pediatric rectosigmoid biopsies were collected from control or patients with ulcerative colitis. Tissue was analyzed for NAT and myeloperoxidase activity.

Results: Dextran sulfate sodium-induced colitis was of similar severity in both NAT WT and knockout mice, and NAT activity was significantly decreased in NAT WT mice. In the setting of colitis, 5-ASA significantly restored colon length and decreased myeloperoxidase activity in NAT knockout but not in WT mice. Myeloperoxidase activity negatively correlated with NAT activity in pediatric patients, but correlation was not observed in adult patients.

Conclusions: Inflammation decreases NAT activity in the colon of mice and human pediatric patients. Decreased NAT activity enhances the therapeutic effect of 5-ASA in mice. A NAT activity assay could be useful to help predict the efficacy of 5-ASA therapy.

FIGURE 1

Appearance of colonic mucosa and 5-ASA metabolism in Nat1/2 KO mice. (A) Representative images of hematoxylin and eosin stained distal colon in Nat1/2 WT and KO mice. Low resolution (4x) and high resolution (10x) of area indicated by rectangle in low resolution image. Bar = 0.2 mm (B) Representative traces of NAT enzyme activity show fluorescence (Ac-5-ASA: 312 nm excitation/ 437 nm emission) over 120 seconds of Nat1/2 WT (●) or Nat1/2 KO (□) mice distal colon homogenate immediately after the addition of 5-ASA and AcCoA.

FIGURE 2

Effect of 5-ASA on DSS-induced colitis in Nat1/2 WT and KO mice. DSS colitis was induced by adding 3.5% DSS to drinking water for 5 days followed by regular drinking water for two days. During this 7 day DSS and regular water treatment, mice were gavaged daily with vehicle (Veh) or 75 mg/kg/day 5-ASA (ASA). Control (Con) mice did not receive DSS or any treatment. (A) Whole colon lengths of excised mouse colon at day 7. Values are mean ± SEM, **P < 0.01, ***P < 0.001 vs. corresponding control, #P < 0.05 Nat1/2 KO Veh vs. Nat1/2 KO ASA. (B) Percent baseline weight changes at day zero (all mice at 100 %) and day 7. **P < 0.01, ***P < 0.001 vs. corresponding control. (C) MPO activity of distal colon in response to DSS. Values are mean ± SEM. *P < 0.05, **P < 0.01 vs. corresponding control; #P < 0.05 Nat1/2 KO Veh vs. Nat1/2 KO ASA. (D) NAT enzyme activity of distal colon. Values are mean ± SEM. ***P < 0.001 vs. control. (E) Correlation of NAT versus MPO enzyme activity of WT control (○) with no DSS, and vehicle treated (), or ASA treated (◆) during DSS. Each point represents one mouse. Linear regression with 95 % confidence levels (dotted line).

FIGURE 3

Measurement of human NAT activity. (A) Representative traces of NAT enzyme activity at 312 nm excitation and 437 nm emission. Fluorescence of colon homogenate (x) was recorded over 120 seconds, then 5-ASA (ASA) was added sequentially to the same homogenate (o), then thirdly, AcCoA was added to start the reaction (●). After 5-ASA or AcCoA addition, representative traces were reset to time zero for comparison. (B) Colon homogenate with 5-ASA (○) compared to the spectra of 5-ASA standard (dotted line). Same colon homogenate plus 5-ASA 30 minutes after addition of AcCoA (●), to initiate the reaction, compared to the spectra of Ac-5-ASA standard (dot-dash-dot line).

FIGURE 4

Effect of inflammation on 5-ASA metabolism in pediatric rectosigmoid biopsies. (A) MPO enzyme activity (B) and NAT enzyme activity was measured in rectosigmoid biopsies of de-identified pediatric control and ulcerative colitis (UC) patients. Values are mean ± SEM. *P < 0.05 and ***P < 0.0001 vs. corresponding control. Pediatric control patients are assigned the letters #a, through h, solid colored circles; ulcerative colitis patients are #i and j, solid colored squares. Graphs (C and D) show the correlation of NAT to MPO enzyme activity and are analyzed by linear regression with 95 % confidence levels (dotted line). (C) Each point represents one patient. Values are mean ± SEM for MPO and NAT activity. The region between 0 and 200 μmol H₂O₂/min/mg MPO activity is expanded to better show the SEM for each patient. (D) Each point represents one biopsy and each color (solid colored symbol) represents one patient.

FIGURE 5

Effect of inflammation on 5-ASA metabolism in adult rectosigmoid biopsies. (A) MPO enzyme activity (B) and NAT enzyme activity was measured in rectosigmoid biopsies of de-identified adult control and ulcerative colitis (UC) patients. Values are mean ± SEM. ***P < 0.0001, vs. corresponding control. NAT activity between control and UC biopsies are not significant. Adult control patients are assigned the letters #k, through p, solid colored circles; ulcerative colitis patients are #q and r, solid colored squares. Graphs (C and D) show the correlation of NAT enzyme to MPO enzyme activity and are analyzed by linear regression with 95 % confidence levels (dotted line). (C) Each point represents one patient. Values are mean ± SEM for MPO and NAT activity. The region between 0 and 200 μmol H₂O₂/min/mg MPO activity is expanded to better show the SEM for each patient. (D) Each point represents one biopsy and each color (solid colored symbol) represents one patient.