REMEMBER THOSE slow, boring mornings when after reading over breakfast that 25 cents and the box top would get us a decoder ring, we dawdled to the point of learning that certain minerals were present in our cereal--though not too much, just a trace? In those days a trace meant less than one part per million; nowadays, scientific refinement allows measurement to parts per billion.
Wayne R. Wolf, a research chemist at the Nutrient Composition Lab in USDA's Beltsville Agricultural Research Center, has been involved in this advance from millions to billions, from the merely incomprehensible to the truly mind boggling. Just how small is a one-billionth part of something?
"One part per billion is a drop of vermouth in a tank car full of gin" says one of Wolf's colleagues. Another figures that about one billion seconds ago Harry Truman was president. However one wants to visualize it, one part per billion is a minuscule quantity. And yet nutrition scientists, using new techniques, are discovering that some minerals--salenium, chromium, zinc, for example--occur in just such minuscule amounts and are required in the optimal human diet.
Along with the advance in technique and knowledge has come a generation of new words such as ultra-trace and vitamer. And, of course, names for new instruments such as the atomic absorption spectrophotometer and liquid chromatograph. The words and the equipment both reflect the inquiry into smaller and smaller quantities, finer and finer discriminations, down into the very atomic structure of the stuff that we call food.
This has led researchers to discover yet another new word, bio-availability, or the interaction of different components of food as they affect the body's ability to absorb and put nutrients to use. Optimal nutrition cannot be described simply as the quantities of nutrients ingested, because the way those nutrients interact influences the food value the body gains from them.
"We are starting to understand that we can't just talk about iron, for example, but rather about different places in the body that iron is used. We have to understand the interactions," says Wolf. "Vitamin C intake enhances iron availability. The iron in certain grains and cereals is not as available as the iron in red meat. So if you eat a breakfast with cereal only, you are not getting as much iron as you would if you had orange juice or meat with it. It really is a complex situation. You can maximize a diet as far as iron goes, but what are you doing to the other elements?"
Down the hall from Wolf, research chemist Joseph Vanderslice is asking similar questions, but he's concerned with vitamins, in particular vitamin B-6. There are six forms of vitamin B-6, all close to one another in chemical structure, but not all occurring in the same proportion in different foods, and not all as readily absorbed by the human digestive system. These six are vitamers, or variable forms within a single vitamin and they complicate the research in several different vitamins.
All B-6 vitamers function identically in nutrition if broken down and put to use by the body, but it appears that not all are equally available to the human metabolic system. "If you can get any of the B-6 vitamers out of the intestines into the body, they have the same activity. But the question of which you can get out of the intestinal wall best is still up for grabs. Up to five years ago we had no way to distinguish the forms of B-6. The big technique that has now made a difference has been chromatography, both gas and liquid," says Vanderslice.Wolf, who concentrates on inorganic elements in food--what we call minerals--glances up at the index card taped above his desk. On it is written Webster's definition of trace: "an amount of a chemical constituent not quantitatively determined because of minuteness."
"Historically," says Wolf, "trace meant anything below one part per million. But now we can actually measure sub-parts per billion. When we can measure parts per billion, it's no longer a trace. Now people are talking about ultra-trace levels--ultra-trace meaning down around parts per billion, as in the case of chromium. But that term, ultra-trace, drives me up the wall. We get into this terminology trap because we accept the word trace."
Prime among the sophisticated instruments developed to measure such incredibly small quantities of these inorganic elements is the atomic absorption spectrophotometer. It operates on the principle that every element, when excited by a high-energy light source, gives off a discrete and unique wavelength of light or energy.
The procedure involves first removing all organic material from the food being tested--a gram or less of white bread, fresh pork loin, in-season apples, or franchise fried chicken. "We take the food and put it through ashing in a muffled furnace or digestion in strong acids," says Wolf. "We destroy the organic material, which is what you look at and call food, comprised of oxygen, carbon, and hydrogen. Then we look at what's left." Such procedures have to take place in a sterile room, complete with air-filter hoods, since particulate matter floating through the air can measurably increase mineral content.
After ashing, the food sample looks like water sitting in a miniature flask. A plastic tube a fraction of an inch in diameter pulls the sample up into an aspirator, which turns the sample into a fine mist. The misted sample is shot up through a gas burner about three inches long, where it breaks up into individual atoms. At that moment, a high-intensity light bulb flashes into the flame, and the amount of light that emerges on the other side of the flamed sample is measured. The technician knows the wavelength of light being blasted upon the sample in question and the machine calculates the amount and specific wavelength of light that emerges on the other side of the sample. The more light given off at that wavelength, the more of the element within the sample.
Atomic absorption spectrophotometers have been the primary method of mineral analysis in food since the early '60s, but one drawback has been the difficulty in measuring different elements within the same sample. Not only do most machines search for only one wavelength at a time, but the process also destroys the sample, so one could never recalibrate the machine and look at the same sample again. Wolf and his associates have been working on a glorified version of the atomic absorption spectrophotometer, one that can measure up to 20 elements in a single sample, all within a brief 30-second blast.
"When you get into multi-element situations, it's much easier to get into errors because you're measuring so many more things," says Wolf. "In a single-element study you optimize for each element. Any multi-element technique is a compromise, and it takes a while to understand where the compromises are. It increases the analytical headaches, but it has great potential."
Atomic absorbtion spectophotometry, which separates one element from another, would not work with vitamins, which are organic compounds made up primarily of different combinations of the same three elements--carbon, hydrogen and oxygen. Instead, researchers such as Vanderslice use chromatography, a method that makes fine distinctions between closely resemblant chemicals by discriminating between their reactions to a controlled chemical or physical environment.
When Vanderslice wants to determine the relative proportions of two vitamers of B-6 in a sample of food, he homogenizes his sample and, taking care not to let it age or oxidize, introduces it into a thin vertical column, about 6 inches long, filled with a polymer resin specially manufactured to test certain chemical characteristics. He uses liquid chromatography, pushing the sample through a column in liquid form. Other researchers, interested in other vitamins, use gas chromatography, in which the sample is vaporized as it enters the column.
One of the vitamers may have a stronger negative charge than another, and to separate them a positively charged polymer is used. As the food sample travels down the column of polymer, the vitamer with the stronger negative charge sticks onto the polymer more immediately and more forcefully than the other. The vitamer with the weaker charge slides on through more quickly. When they come out the other end of the column, the two vitamers emerge in two surges, and detectors record the difference between them. "You inject a slug of the unknown into this stream," says Vanderslice. "Different components will elute at different points."
"It's like touching a pen to a piece of filter paper," explains Gary Beecher, director of the Nutrient Composition Lab, who uses liquid chromatography to test beta-carotene, one of several hundred vitamers of vitamin A. "If you would take a pen and make a spot on a piece of paper, then put a drop of methanol or ethanol on that spot, the color would start to move outward and it would make four different rings of color, representing four different components in the ink."
A similar separation occurs within the column of the liquid or gas chromatograph, and the pattern of the slight differences between the vitamers is recorded as a pattern of electrical signals by a microcomputer attached to the end of the column. Vanderslice's raw data appear as chromatograms: computer-printed graphs of peaks and dips, each peak indicating the relative amount of vitamer present in the food sample being tested.
"The biggest job is getting the vitamin away from the complex matrix that we call foods," says Vanderslice. "B-6 is often bound to proteins. The easiest foods we have tested were muscle tissue from meats , but the tougher problems were manufactured foods, because they actually put in binders to hold things together."
Vanderslice has compared the forms of vitamin B-6 found in foods naturally with those found in processed or manufactured foods. He compared human breast milk with baby formulas, for example, and found that despite the manufacturers' efforts to fortify formulas with B-6, "the form of B-6 that you find in mother's milk is completely different from the form found in formulas."
More information than ever before is available about the value of foods. "We're in a very interesting period in our society," says Beecher, just back from a National Nutritional Data Bank Conference in Minneapolis. "It's the beginning of the information age. This is kind of mind boggling for many scientists, and it's important that we keep track of the quality of the information that we generate. The scientists that are studying metabolic changes as a result of changing the diet need to have a very accurate, very precise measurement of what the person is eating. They need to know not only the concentration of B-6 but also the forms of the vitamins.
"I think what we're seeing is a change in the orientation of nutrition in this country. Up until some years ago, we were concerned primarily with deficiency--providing enough nutrients in the diet to prevent deficiency diseases. There are still some pockets of deficiency diseases in the United States, but by and large the philosophy has changed to providing nutrients for optimal human nutrition--that is, enough nutrients for the body so that over the long run it can run at its optimal level, able to resist disease and infection, able to adapt to stress.
"We know that nutrition has made an important difference in the area of cardiovascular disease. We have a pretty good idea that lowering fat and reducing cholesterol levels helps in maintaining cardiovascular health in the human population. I think we have good reason to expect the same results in cancer. We're not going to cure it, but we can prevent it," says Beecher.
And, even if the reasons are not yet understood, it's the tiny amounts of zinc, chromium, selenium, beta-carotene and other elements that seem to make a difference, though not in themselves. Taking zinc or selenium tablets does not replicate the effect of ingesting these elements along with other food components in a balanced diet. An excess of the so-called ultra-trace minerals can be as harmful as a deficiency, according to the experts. Synthetic vitamins may not include the same vitamers as a natural source, and good nutrition, they say, depends upon nutrient bio-availability.