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Human intestinal fatty acid binding protein in peritoneal fluid is a marker of intestinal ischemia
Human Intestinal Fatty Acid Binding Protein in Peritoneal Fluid is a Marker of Intestinal Ischemia R. Sonnino, G. Ereso, J. Arcuni, and R. Franson
I
NTESTINAL ischemia is a surgical emergency for which there is no accepted biochemical marker. Intestinal fatty acid binding protein (I-FABP) is a 15-kD protein that is uniquely located at the tips of the intestinal mucosal villi.1 It constitutes approximately 2% to 3% of the protein of the enterocyte and is normally undetectable in the peripheral circulation.2 Experimental studies by radioimmunoassay have shown that serum I-FABP is a sensitive and organspecific biochemical marker for intestinal mucosal injury in experimental models of mesenteric ischemia2 and in premature infants with NEC.3 Serum I-FABP has also been used to monitor an intestinal transplant graft in a syngeneic human transplant recipient, and in the systemic inflammatory response system (SIRS).4 The purpose of this study was to determine whether human I-FABP (hI-FABP) could be measured in peritoneal fluid and whether it could reliably predict the presence of intestinal ischemia using a newly developed competition ELISA. METHODS Sample Collection With IRB approval, random samples of peritoneal fluid were collected from pediatric and adult patients with either no bowel pathology (n ⫽ 11) or intestinal ischemia of variable degrees (n ⫽ 5). Pathologic findings in these patients included incarcerated bowel (1), appendicitis (1), ischemic bowel (2), and frankly necrotic bowel (1).
ies were 99% and 95%, respectively. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The standard curve was linear between 2.7 ng/mL and 1000 ng/mL (r ⫽ .97). hI-FABP levels in patients without intestinal pathology were ⬍2.71 ng/mL (the limit of detection of our assay), while in patients with intestinal pathology they averaged 155.17 ⫾ 94.43 ng/mL (range 20.42 to 527.74). The difference was highly significant (P ⬍ .0025).
CONCLUSIONS
These findings demonstrate the presence of hI-FABP as a marker of injured bowel in peritoneal fluid, and strongly suggest a positive correlation between hI-FABP levels and degree of intestinal ischemia. Although no correlation with outcome has been made in this study, the accessibility of these fluids in patients being evaluated for intestinal pathology may significantly enhance early detection of intestinal ischemia and improve early treatment of the underlying pathology before the onset of irreversible injury. Because a qualitative version of the assay may be shortened considerably to meet clinical needs, this ELISA may offer a method of early detection of intestinal ischemia, including technical complications and rejection in intestinal transplants, leading to treatment of the underlying pathology before the onset of irreversible injury.
ELISA The assay was developed using specific, affinity-purified antibodies raised against purified recombinant hI-FABP in male New Zealand White rabbits. Microtiter plates were coated overnight at 4°C with purified hI-FABP, and blocked with 5% milk protein in isotonic saline. The samples or hI-FABP standards were pre-incubated for 30 minutes with an equal volume of antibody. The plates were decanted and reaction mixture (sample ⫹ antibody) was added to the wells and incubated for 90 minutes. The wells were washed, the secondary antibody was added and allowed to incubate for 90 minutes. After washing again, substrate (TMB) was added and incubated for 20 minutes. The reaction was stopped and the absorbance was read at 450 nm.
RESULTS
The ELISA showed no cross-reactivity with other serum or peritoneal fluid components. The mean analytical recover0041-1345/00/$–see front matter PII S0041-1345(00)01225-2 1280
REFERENCES 1. Ockner RK, Manning JA: J Clin Invest 54:326, 1974 2. Gollin G, Marks C, Marks WH: Surgery, 113:545, 1993 3. Edelson MB, Rozycki HJ, Bagwell CE, et al: Ped Research 39:206A, 1996 4. Kuo PC, Morris J, Marks WH, et al: Clin Transplant 10:282, 1996
From The University of Kansas SOM, Kansas City, Kansas, and The Medical College of Virginia, Richmond, Virginia. Supported in part by grant #6-FY-98-0649 from the March of Dimes Birth Defects Foundation. Address reprint requests to Dr R.E. Sonnino, 3032 Delp, 3901 Rainbow Blvd, Kansas City, KS 66160. © 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 32, 1280 (2000)
I
NTESTINAL ischemia is a surgical emergency for which there is no accepted biochemical marker. Intestinal fatty acid binding protein (I-FABP) is a 15-kD protein that is uniquely located at the tips of the intestinal mucosal villi.1 It constitutes approximately 2% to 3% of the protein of the enterocyte and is normally undetectable in the peripheral circulation.2 Experimental studies by radioimmunoassay have shown that serum I-FABP is a sensitive and organspecific biochemical marker for intestinal mucosal injury in experimental models of mesenteric ischemia2 and in premature infants with NEC.3 Serum I-FABP has also been used to monitor an intestinal transplant graft in a syngeneic human transplant recipient, and in the systemic inflammatory response system (SIRS).4 The purpose of this study was to determine whether human I-FABP (hI-FABP) could be measured in peritoneal fluid and whether it could reliably predict the presence of intestinal ischemia using a newly developed competition ELISA. METHODS Sample Collection With IRB approval, random samples of peritoneal fluid were collected from pediatric and adult patients with either no bowel pathology (n ⫽ 11) or intestinal ischemia of variable degrees (n ⫽ 5). Pathologic findings in these patients included incarcerated bowel (1), appendicitis (1), ischemic bowel (2), and frankly necrotic bowel (1).
ies were 99% and 95%, respectively. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The standard curve was linear between 2.7 ng/mL and 1000 ng/mL (r ⫽ .97). hI-FABP levels in patients without intestinal pathology were ⬍2.71 ng/mL (the limit of detection of our assay), while in patients with intestinal pathology they averaged 155.17 ⫾ 94.43 ng/mL (range 20.42 to 527.74). The difference was highly significant (P ⬍ .0025).
CONCLUSIONS
These findings demonstrate the presence of hI-FABP as a marker of injured bowel in peritoneal fluid, and strongly suggest a positive correlation between hI-FABP levels and degree of intestinal ischemia. Although no correlation with outcome has been made in this study, the accessibility of these fluids in patients being evaluated for intestinal pathology may significantly enhance early detection of intestinal ischemia and improve early treatment of the underlying pathology before the onset of irreversible injury. Because a qualitative version of the assay may be shortened considerably to meet clinical needs, this ELISA may offer a method of early detection of intestinal ischemia, including technical complications and rejection in intestinal transplants, leading to treatment of the underlying pathology before the onset of irreversible injury.
ELISA The assay was developed using specific, affinity-purified antibodies raised against purified recombinant hI-FABP in male New Zealand White rabbits. Microtiter plates were coated overnight at 4°C with purified hI-FABP, and blocked with 5% milk protein in isotonic saline. The samples or hI-FABP standards were pre-incubated for 30 minutes with an equal volume of antibody. The plates were decanted and reaction mixture (sample ⫹ antibody) was added to the wells and incubated for 90 minutes. The wells were washed, the secondary antibody was added and allowed to incubate for 90 minutes. After washing again, substrate (TMB) was added and incubated for 20 minutes. The reaction was stopped and the absorbance was read at 450 nm.
RESULTS
The ELISA showed no cross-reactivity with other serum or peritoneal fluid components. The mean analytical recover0041-1345/00/$–see front matter PII S0041-1345(00)01225-2 1280
REFERENCES 1. Ockner RK, Manning JA: J Clin Invest 54:326, 1974 2. Gollin G, Marks C, Marks WH: Surgery, 113:545, 1993 3. Edelson MB, Rozycki HJ, Bagwell CE, et al: Ped Research 39:206A, 1996 4. Kuo PC, Morris J, Marks WH, et al: Clin Transplant 10:282, 1996
From The University of Kansas SOM, Kansas City, Kansas, and The Medical College of Virginia, Richmond, Virginia. Supported in part by grant #6-FY-98-0649 from the March of Dimes Birth Defects Foundation. Address reprint requests to Dr R.E. Sonnino, 3032 Delp, 3901 Rainbow Blvd, Kansas City, KS 66160. © 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 32, 1280 (2000)