Source: PURDUE UNIVERSITY submitted to
ELUCIDATION OF THE CAUSE OF PORCINE HEMORRHAGIC SYNDROME
 
PROJECT DIRECTOR: Hooser, S. B. Andrews, D. A. Christian, J. A.
 
PERFORMING ORGANIZATION
VETERINARY PATHOBIOLOGY
PURDUE UNIVERSITY
WEST LAFAYETTE,IN 47907
 
NON TECHNICAL SUMMARY: Porcine hemorrhagic syndrome (PHS)is a feed-related bleeding syndrome of pigs. The goal of this project is to identify and eliminate the cause of PHS. Identification will allow for testing of feeds for this agent thus eliminating the need to supplement feeds with menadione to prevent PHS. Once the cause is identified, it may be possible to prevent its formation in swine feeds.
 
OBJECTIVES: Objectives: The goal of this project is to elucidate the cause of porcine hemorrhagic syndrome (PHS). Our hypothesis is that an unknown vitamin K antagonist which inhibits VKOR is sometimes present in pig feed resulting in coagulopathies in the affected swine. We will test this hypothesis through the following Objectives: 1. To develop an in vitro test for inhibition of vitamin K epoxide reductase. Clinically, the bleeding syndrome in pigs with PHS is known to respond favorably to the administration of vitamin K which is used as the antidote for poisoning with anticoagulant rodenticides. It is also known that the mechanism of action of anticoagulant rodenticides is to inhibit the enzyme, vitamin K epoxide reductase (VKOR) in the liver. This suggests that the causative agent of PHS acts by inhibition of VKOR. For this objective, hepatic microsomes containing active VKOR will be isolated from pigs to determine the activity of this enzyme in vitro. Once developed, this assay will then be used to screen compounds isolated from pig feed for inhibition of VKOR activity. 2. To isolate compounds with anticoagulant properties from pig feed. In one case of PHS, and in several feed samples, we have identified an unknown compound(s) with UV spectral characteristics similar to anticoagulant rodenticides. Using HPLC, we will isolate and purify this compound to the greatest degree possible. We will then compare it to other known anticoagulants for its ability to inhibit VKOR activity in porcine hepatic microsomes. 3. To identify the chemical structure of compounds in pig feed which inhibit VKOR. Any compounds found in pig feed which inhibit VKOR activity will be subjected to mass spectroscopy and nuclear magnetic resonance (NMR) for structural elucidation. 4. To identify the unknown anticoagulant in clinical cases of PHS. With the help of Indiana swine practitioners, we will attempt to identify outbreaks of PHS as they occur. When these cases are identified, we will coordinate with the referring veterinarians and owners to collect blood from the affected pigs for determination of blood coagulation times (PT and PTT), hematology and serum chemistries. In addition, we will test the blood and feed for possible anticoagulant compounds including the unknown compound that we have previously identified.
 
APPROACH: Objective 1: To develop an in vitro test for inhibition of vitamin K epoxide reductase. Briefly, liver samples will be collected from pigs at slaughter (see animal use statement below) and immediately frozen in liquid nitrogen. The liver samples will be taken from pigs being slaughtered for consumption. No pigs will be killed specifically for this project. The frozen liver samples will be homogenized and subjected to differential centrifugation to prepare and purify the microsomes (endoplasmic reticulum). These microsomes will then be used in an assay for vitamin K epoxide reductase activity. VKOR assay: hepatic microsomes will placed in buffer and heated to 37oC. The reaction will be initiated by the addition of dithiothreitol (DTT, the reducing agent) to the reaction vessel. The reaction will be stopped by the addition of 1ml of isopropanol : hexane (3:2). The reaction mixture will then be extracted with hexane and the reduction of vitamin K epoxide to vitamin K will be quantified by HPLC with UV detection. Objective 2: To isolate compounds with anticoagulant properties from pig feed. Compounds extracted from pig feeds and tissues with UV spectral characteristics similar to anticoagulant rodenticides will be isolated by collection of the HPLC fractions which contain them. The amount will be roughly calculated based on comparison to purified standards of known anticoagulant rodenticides. These compounds will then be evaluated in the microsomal VKOR assay and their inhibitory activity compared to DMSO (control) and standards of anticoagulant rodenticides. Objective 3. To identify the chemical structure of compounds in pig feed which inhibit VKOR. Isolated and purified compounds which inhibit VKOR activity will be structurally analyzed by HPLC with detection by mass spectroscopy. Purified HPLC fractions containing the compounds will be further analyzed by nuclear magnetic resonance (NMR). Both mass spectroscopy and NMR will be performed using the services of the mass spectroscopy laboratory at Purdue University. Analysis via mass spectroscopy and NMR will produce detailed information on the structure of the unknown compound(s). Objective 4. To identify the unknown anticoagulant in clinical cases of PHS. With the help of Indiana swine practitioners, we will attempt to identify outbreaks of PHS as they occur. When these cases are identified, we will coordinate with the referring veterinarians and owners to collect whole blood, EDTA blood and citrated blood from the clinically affected pigs. Blood, serum and citrated plasma from the PHS suspect pigs will receive a complete blood count (CBC), chemistry profile, and coagulation profile (PT, PTT, platelet count, fibrinogen and fibrinogen degradation products (FDP)) to assure that no concurrent disease processes (such as liver disease) are responsible for the bleeding syndrome. In addition, we will test the blood and feed by HPLC for possible anticoagulant compounds including the unknown compound that we have previously identified. If a suspect compound is identified by HPLC, then it will be isolated and its ability to inhibit VKOR in the microsomal assay will be determined.
 
CRIS NUMBER: 0189396 SUBFILE: CRIS
PROJECT NUMBER: IND073068 SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: May 1, 2001 TERMINATION DATE: Sep 30, 2005

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
314351011504.2100%

CLASSIFICATION HEADINGS
KA314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals
S3510 - Swine, live animal
F1150 - Toxicology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks


RESEARCH EFFORT CATEGORIES
BASIC (N/A)%
APPLIED 100%
DEVELOPMENTAL (N/A)%

KEYWORDS: veterinary medicine; swine; livestock production; pigs; feed; vitamin k; hemorrhagic diseases; toxicology; animal health; feed contamination; feed quality; reductases; enzyme inhibitors; mechanism of action; enzyme activity; microsomes; coagulation; hplc (chromatography); mass spectroscopy; nuclear magnetic resonance; disease outbreaks; epoxides; liver; tissue analysis; quantitative analysis; purification; chemical analysis; structural analysis

PROGRESS: May 1, 2001 TO Sep 30, 2005
An in vitro assay for vitamin K epoxide reductase (VKOR) activity has been developed and validated in our laboratory for swine, bovine, equine, canine, rat, mouse, and human hepatic microsomes. We believe that VKOR activity is inhibited by the agent which causes Porcine Hemorrhagic Syndrome (PHS). We have tested small quantities of extracts from feed and the livers of pigs with bleeding disorders, compared these to known anticoagulants such as Warfarin, and found that they all do inhibit VKOR. We have also compared VKOR activity in pig liver to that in the livers of other species and found that they are similar. These results have been published.

IMPACT: 2001-05-01 TO 2005-09-30 We hope that the information gained from this study will help the swine industry to prevent the occurrence of porcine hemorrhagic syndrome and eventually eliminate the need to supplement swine feed with vitamin K.

PUBLICATION INFORMATION: 2001-05-01 TO 2005-09-30
Wilson CR, Sauer, J-M, Carlson, GP, Wallin R, Ward MP, Hooser SB. Species comparison of vitamin K1 2,3-epoxide reductase activity in vitro: kinetics and warfarin inhibition. Toxicology 189: 191-198, 2003.

PROJECT CONTACT INFORMATION
NAME: Hooser, S. B.
PHONE: 494-7440
FAX: 494-9181