ChemMatters December 1988 Page 4

© Copyright 1988, American Chemical Society

MYSTERY MATTERS

Saving Arnold

by Michael W. McClure

John Evans had been in the hog business for many years and thought he knew everything about raising pigs. But one cold, rainy day last November he was confronted by something mysterious—and deadly. The Evans hog farm is a family enterprise. John’s wife, Kathy, does the bookkeeping and their daughter, Ellie, helps with the feeding. Ellie especially likes caring for the baby pigs, and she even convinced her dad that the family should adopt a piglet as a pet. They named him Arnold.

Too quiet

John and Ellie were making their evening rounds through the furrowing house, the nursery barn for sows and new piglets, when they sensed that something was wrong. The furrowing house is usually a noisy place filled with squeals and grunts, but today it was quiet—too quiet. Ellie rushed down the narrow aisle between the stalls, stopped, stared into a stall, and screamed. Four piglets lay motionless in the straw. They were dead. A hurried check of the other stalls revealed that 26 piglets were dead, and many more were sick. John immediately called the veterinarian. By the time she arrived, it was apparent to John that an illness he had never seen before was affecting nearly all of his hogs. Even Arnold was listless and had no appetite. John and Ellie knew it would be a long night.

By morning the situation had grown more desperate; 10 more piglets had died. By now the veterinarian had examined dozens of pigs and noted the symptoms: shivering, tremors, weakness, and hemorrhaging. Her examinations had ruled out infections that were viral, bacterial, or parasitic. This left poisoning as a possibility, and she suggested that the hogs may have ingested some pesticide. John argued that he had always taken extra precautions with pesticides, and he had a firm rule that no man-made chemicals were ever allowed in the furrowing house, only pig feed. Something about the word "feed" triggered John’s memory—a new batch of feed had arrived only two days before the symptoms appeared. Could the feed be contaminated? Together, they quickly scooped pig feed into some large paper bags.

Poison search

It was mid-afternoon when the veterinarian stalked into my office with four bags in her arms and quickly explained the situation. I looked closely at the feed. It appeared normal. The symptoms could be caused by a number of poisons. Lead poisoning affects the central nervous system and can cause tremors; some commercial rat poisons cause hemorrhaging—there were at least a dozen possibilities. A thorough chemical analysis could take weeks, but I knew I would have to act quickly if we were to save the remaining animals.

Suddenly I thought, "What if the feed was not contaminated by man-made chemicals? What if there was a natural contaminant present?" With the proper conditions of moisture and temperature, mildews and molds can invade almost any food (such as a slice of bread left uncovered for a few days), and some fungi excrete deadly poisons. Aspergillus flavus, a green powdery mold, is one of the worst, producing noxious chemicals called aflatoxins. Certainly the pig feed did not look moldy, but the process of grinding and blending can mask the green spores of Aspergillus. It was only a hunch, but we had to try something immediately.

Hog feed is a blend of ground corn, minerals, vitamins, and steroids, and the corn contains numerous oils, carbohydrates, and proteins. Isolating and identifying a tiny amount of aflatoxin from such a mixture is a challenging task. Fortunately there are separation techniques that can remove most of these substances. I elected to use a method based on solvent extraction and column chromatography. First, I ground some of the feed to a fine, dusty powder and mixed it thoroughly to make it uniform. Then, in an ordinary kitchen blender, I blended 50 g of the powder with 100 ml of a water-methanol mixture. The resulting sloppy mixture contained insoluble minerals and fiber, which were removed by filtering.

After the pig feed was blended with water, the water solution was placed in a separatory funnel with a few milliliters of toluene. During vigorous shaking, nonpolar compounds dissolved in the toluene, while polar compounds remained in the water. (Water is polar because its molecules have separated, partial electric charges). When the shaking stopped, the toluene formed a later on top of the water, thereby separating the nonpolar compounds from the polar ones.

Then I transferred the liquid to a separatory funnel, added a few milliliters of toluene, shook the stoppered funnel for one minute, and then let it stand. Toluene is immiscible in the water-methanol mixture, and a layer of toluene soon separated from the water-methanol layer—like oil separating from vinegar in a salad dressing. Most of the substances in the feed—carbohydrates, proteins, some vitamins—remained in the water layer. Fat-like substances such as aflatoxin moved into the toluene layer. I discarded the water layer and began preparing a column.

The chromatography column used to test for aflatoxin is packed with several layers of porous compounds. The test solution and selected solvents are poured into the top and allowed to drain through the column. The different substances in the liquid adhere to different layers of the column effectively separation the dissolved substances. Aflatoxin adheres to the top of the magnesium silicate layer and fluoresces blue when exposed to ultraviolet light. This technique can detect 1 part aflatoxin in 100 million parts of feed.

Mobilizing the solution

In column chromatography, a glass tube is packed with a solid adsorbent material, called the stationary phase. The mixture to be separated—the mobile phase—is poured into the top of the column. As the mobile phase diffuses downward through the stationary phase, the dissolved molecules stick to the porous solid. Next a solvent is poured into the column, and this is often—although not in the aflatoxin test—followed by other solvents, in order of increasing polarity. Molecules that are soluble in the first solvent become unstuck from the stationary phase and travel down the column. As the solvent polarity changes, other molecules let go of the stationary phase and move gradually down the column. The original sample is a mixture of many substances with different solubilities. Because they move down the column at different rates, the mixture becomes separated.

I used a glass tube only 15-cm long and packed it with layers of calcium sulfate, alumina, silica gel, and magnesium silicate. I added 1 ml of the toluene solution and let it drain into the column. Next I added a few milliliters of a chloroform-acetone mixture, then let the column drain for ten minutes. If aflatoxin were present, it should migrate to the magnesium silicate layer, but it would still be invisible because the aflatoxin occurs in minute amounts and is colorless.

Closing in

The sample workup and column separation had taken only 30 minutes. The veterinarian and I took the column to a darkened room, covered our eyes with protective goggles, and switched on an ultraviolet lamp. A soft, eerie blue-green ring radiated from the boundary between the silica gel and magnesium silicate layers. Aflatoxin! My hunch was right. Though aflatoxin is normally invisible, its molecular structure causes it to fluoresce when exposed to UV light.

The veterinarian rushed from the lab to call John. She prescribed vitamin K for the sows and piglets to control the hemorrhaging, and told John to replace the contaminated feed with clean, fresh corn. Fortunately, the contaminated batch of feed had not yet been used everywhere on the farm, and several hundred older hogs were not affected. The pigs in the furrowing house began a slow recovery. A few days later John and Ellie were finally able to smile again—John because most of his hogs had been saved, and Ellie because Arnold was eating like a pig again.

BIOGRAPHY

Michael W. McClure worked for several years as a chemist at a state veterinary diagnostic laboratory, where he investigated cases of poisoning in pets and livestock. He now teaches chemistry at Hopkinsville Community College, Hopkinsville, Ky.

REFERENCES

McClure, M. "Aflatoxins in Corn," Diagnostic News 1980, 2, 4, Murray State University Veterinary Diagnostic Center, Ky.

Nordheim, A. et al. "Inhibition of Salt-Induced Conversion of B-DNA to Z-DNA by Aflatoxin B1," Science 1983, 219, 1434.