Wisconsin Public Television University Place  How Vitamin Research Uncovered Essential Minerals  Roger Sunde  6/19/13 UP888  cc >> Good evening, everyone. I'm Cassie Immel from the UW Biotech Center. This evening we have Dr. Roger Sunde. He's a professor of Nutritional Sciences at the UW. He earned his PhD from the University of Wisconsin in biochemistry, and since then he's been an NIH Postdoc Fellow at the Rowett Research Institute in Scotland, the assistant and associate professor at the University of Arizona, and the Food for the 21st Century Nutrition Cluster Leader, Professor and Chair of Nutritional Sciences, and Professor of Biochemistry at the University of Missouri. Then, in January 2004, he returned to the greatest university on Earth to be Professor and Chair of the Department of Nutritional Sciences. Currently, he does research on molecular selenium nutrition and how the deficiency can effect gene expression, and he'll go into that more tonight. So, without further ado, let's welcome Dr. Sunde. [APPLAUSE]  >> Thank you, Cassie, and thanks to Tom for inviting me. I'm thrilled to be here tonight. 

This is going to give me an opportunity, in this hundredth year anniversary of the discovery of vitamin A, to talk about its impact, both on the research here at the University of Wisconsin. This is foundation, really, for the biological strength and recognition at this university, but it's also the foundation for our current understanding of nutrition. And so I think it's important to make that leap, and I'll connect that then with what I work in, which is trace element biochemistry and nutrition. I also am going to emphasize tonight the use of multiple models to study nutrition. This has been a true strength of this research, and you'll see that again as I mention some species in passing and in detail, and it's one that's very important today at the USDA and maybe up in Ag Hall. They're starting to think now that you can only do nutrition research and biological sciences in agricultural animals. And look to see how that foundation is done. You'll see that it's done in the rats and pigeons. It's done in dogs. Okay? It's done in mice. 

It's not done in livestock animals, and this is where the advances really came and we need to continue that. I'm also going to emphasize this concept that was developed here at the University of Wisconsin, the biological method of analysis, and try to make that connection. So let's proceed a little bit then. 125 years ago our understanding of nutrition at that time was based on these three men and their ideas: Liebig, Atwater, and Pasteur. So, Liebig was the researcher, the professor at Giessen in Germany. He's the one who discovered that plants needed nitrogen. He promoted the use of ammonia, phosphorus, potassium as fertilizer. Some say he's the father of fertilizers then. And central to that was chemical analysis of foods to determine what was good and what was bad. So this is looking at the macronutrients. 

So proximate analysis, you may remember, you can take a food or a diet and dry it, and the difference in weight is going to tell you what the water content is. You can burn it, and what's left is ash. That's going to be the mineral content. He used a Kjeldahl digestion which determines the amount of nitrogen in food, and because proteins contain about, on average, 16% nitrogen, you can multiply that by 6.25 and that will tell you how much protein is there. You can also do ether extraction to tell you how much lipid is there. That's the fat fraction. And then what's left then is carbohydrate. Proximate analysis and that was 125 years ago how we could assess diets, whether they were good or bad is how well they matched up with some preconceived ideas about what should be in the diet. Now, a second important item was W.O. Atwater developed respiratory calorimetry where he could determine then the amount of calories that were in each of these types of foods or burn foods completely. And so that's where the 4 kcals per gram for carbohydrate, the 9 kcals per gram for fat, and the 4 kcals per gram for protein were derived. 

So that was a second way that you could analyze for food with this chemical method of analysis. The other big thing that was going on at the turn of the last century was this germ theory with regard to disease. So, from Pasteur the idea was then that these diseases of humans and livestock were largely caused by microorganisms of one sort or another. In fact, Aikman, who was the first person who was studying B1 in beriberi thought that what he was finding in the rice polishings was not a vitamin but instead it was some sort of antimicrobial agent, anti-disease agent that was protecting these animals. So that was the context in research 125 years ago. So Stephen Babcock, this icon here at the University of Wisconsin, famous for more than just Babcock ice cream, developed a test for butterfat that really transformed the dairy land here at the University of Wisconsin. So remember, in that day and age, farmers, if they wanted to get a little bit more money, could put the dirty water from their well or whatever into their milk. They'd have a larger volume and sell it. They'd get a little bit more money. So it was a really harbinger of disease. 

And Babcock derived then the Babcock test where he simply used sulfuric acid, acidified milk, and that destroyed the micelles that hold the fat, the fat would flow up to the top, he could centrifuge that then and determine the percent fat. And suddenly the farmer's adding dirty water to milk, it didn't help him a bit. So he became a true leader then and put America on the map in terms of its dairy product. He also had tremendous stature with the dairymen and the farmers in the state and the legislators, and that certainly helped this university. He was also a little wacky. So he used to tease Atwater and tell him if you use proximate analysis, bituminous coal ought to be a good food. [LAUGHTER] You can ether extract it, get a little bit of oil, a little bit of lipid. You can determine nitrogen and Kjeldahl digestion, multiply that by 6.25 and you've got -- protein. You can burn it. You get some ash, and the rest is carbohydrate. 

It ought to be a good diet. Well, everybody knows that it isn't. [LAUGHTER] But that's the heart then of this next series. So in 1907, Babcock, this really was his second experiment, he talked Carlisle, the chair of animal sciences, into donating 16 cattle then in an experiment where he wanted to formulate diets by proximate analysis that were all identically the same carbohydrate, fat, protein plus some salt. But all that material would come from one plant species: wheat, oats, corn, or a mixture. So he formulated these balanced diets and fed them to cattle. So we hired, from Yale, a young guy who wasn't getting a job, Elmer McCollum who came here then in 1907, and his job was the muscular job of analyzing these diets and then mixing these diets for cows, 16 cattle. You can imagine this guy got some ideas in his head about maybe you could do experiments with smaller animals and less diet. [LAUGHTER] And that's exactly what he did. So the result was then animals who were fed the wheat diet for a couple of years, the cows were stunted and became blind, vitamin A presumably, and the calves were born dead. 

Animals who were fed that oat-based diet were better off. The calves were born alive, but they soon died. But animals on a corn-based diet did extremely well, and their calves did well. And those on the mixed diet did almost as well as on the corn diet. So proximate analysis wasn't telling you how healthy the animals were going to be. It did make a difference. And so that really is the start of these experiments. So McCollum, the muscular guy mixing diets then, read up and devised experiments to do with animals. And there are long stories, and if I was telling you about vitamin A, I could tell you about, he actually had to go down to Chicago and buy the first rats that were brought up here to Madison and became the founder colonies for most experimental animals of this nature in the US. But this as an experiment then was published on June 1, 1913. 

So we fed rats that were diets based on 20%, on casein diets with 20% lard in the minerals that they knew they needed. And they would grow for about eight weeks, and I can show you that right here, probably a poor range of growth, and then they would typically stop. And you can see, if you can see the pointer, there's not much growth. At that point, they could restore growth if they added to the diet eggs or they could add butterfat or they could take an ether extract, something that would take lipids, of eggs and add that. And this diagram simply shows at this point they added ether extract of egg, and suddenly the animals started growing again. There was something that was fat soluble in that product, in butterfat and eggs, that would restore growth. If they extracted the butterfat and shook it with olive oil, which didn't do anything, they also grew. So you could move this from one oil to the next. There was some substance then that allowed growth. And they called that substance vitamine A, named after a Polish scientist, Funk, who was calling these things vital amines. 

So that's a hundred years ago this month. Okay, just 98 years ago, in 1915, McCollum and Davis fed diets and supplemented the rats with vitamin A and also alcoholic extracts, and what they found is that alcoholic extracts from wheat embryo. These would water soluble compounds, and the animals would grow but not from rolled oats. So there was a difference in food product between one and the other, and they concluded that that was the same as this anti-beriberi factor: vitamin B. So this was the B vitamin work that was done here at the University of Wisconsin and published in 1915. A year later, McCollum and Cornelia Kennedy then also devised the idea that there were water soluble substances and fat soluble substances. Okay? And they would prevent a variety of diseases. This work was done in pigeons. So they were actually studying beriberi, just as Aikman had been doing, but they showed that water soluble B would then prevent this neuropathy that otherwise occurs in these pigeons. So here's this bird rat, if you will, that's being used to study a disease like that. 

They also went forward to name these vitamin A, vitamin B, and they suggested the use of those letters then would be a nice way to talk about these things until we understood what they really were, at which time those names would be substituted for that. You can see how well that's done. [LAUGHTER] We know vitamin B1 is thiamine. Vitamin B2 is riboflavin. Vitamin C is ascorbic acid. But pretty soon we talk about vitamin E, most people don't say tocopherol, vitamin D you don't say cholecalciferol or something like that, and so on. So these were key concepts then that were developed in that era. So in the mineral field then, along with these organic substances, this sort of is a summary of what was known in terms of mammals requirements right here. So I can list these six. Calcium, chlorine, iron, phosphorus, potassium, sodium, and iodine had recently been shown first in children at the Cleveland Clinic in Ohio that iodine was required. 

And then plants, obviously we need nitrogen and protein and sulfur. So those substances, those seven or nine substances were the elements that we thought animals needed for growth. So in that contexts then a series of papers, 10 or more papers were published by this group: EB Hart, Steenbock, Elvehjem. You recognize these from some of the libraries and the former art museum. And James Waddell then published this first paper in 1925 entitled Iron in Nutrition: Nutritional Anemia on Whole Milk Diets and the Utilization of Inorganic Iron in Hemoglobin Building. So it was well known that inorganic iron didn't always correct anemia. Sometimes it did, but it wasn't always the case. And so Hart, Steenbock, Elvehjem, and Waddell set out to solve that. So what do you think? Did they do this work in cows? In rats? Pigeons? They started off with rabbits. Okay? So first, a little review. 

We all know red blood cells are red because they contain hemoglobin, and the heme is the colored compound and it's part of this four subunit hemoglobin complex that's involved. And the heme is key weighted by this protoporphyrin nine ring which contains these pyrole rings. So there's an organic component in the iron, and people didn't know very much about the building of iron. And so their hypothesis was, as it says here, because inorganic iron cannot take part in hemoglobin building, it indicates that other building materials might be wanting. So they focused, they thought about this pyrole ring then. Maybe this was an organic vitamin that might be necessary for iron to be part or build hemoglobin. And they had some ideas in their mind, in the back of their mind. They wondered about chlorophyll because it too has these pyrole rings. Or they wondered about vitamin E. Vitamin E had been identified in 1922, and so it was well known. 

It's got rings and so on and so forth. So this was their postulate under those conditions. Now, this is Wednesday Nite at the Lab, and I thought I would take you through some of the old data directly to give you a glimpse of how that really looks. So this is the first table from that 1925 paper. And up on top, in yellow, you can see they actually list the date of this experiment. So these rabbits were fed whole milk diets. So, getting back to milk, it's a pretty good food, so they fed whole milk diets. Okay. For six weeks and what you can see here is you can see the hemoglobin dropped from 83 to 59 or the red blood cells dropped from seven million to four million while the animals gained some weight. But if they supplemented with fresh cabbage, milk plus 50 grams of fresh cabbage a day, you can see the hemoglobin stayed about the same, the weight went up, and so on. 

And for your edification, I've plotted those two panels right here. So the black shows the milk only rabbits, okay, in six weeks, and you can see how they grew a little. But the ones that got cabbage plus milk grew a lot better. You can see that, can't you? Okay. Similarly, red blood cells, the erythrocytes stayed the same, but if they were fed milk only, it dropped. And their hemoglobin levels also dropped if they were fed milk only. Now, I'm going to add the bottom half of that table now to this graph. So they also showed that if they just put inorganic iron into the diet as well, those hemoglobin levels, erythrocyte levels, also fell. Seven to five, nine to eight or so, and so on. But if they put iron in cabbage, that protected. 

So there's something in that cabbage along with the iron that makes the iron available, or something's going on there. If they dried the cabbage and did that, it worked as well. So that didn't interfere Sometimes organic substances, if you dry them, there are problems. So I'm not going to go through all that data, but the next studies in that paper then showed that fresh and dried cabbage contains some iron. So they could have been giving iron along with whatever was in there. Maybe that was doing it. So they resorted to extracts. But when they took alcoholic extracts of cabbage, or they looked at cornmeal, plus iron, they would protect even though these extracts were free of iron. So there was something in addition to iron that was necessary. They also had an alternative strategy, and they kind of swung for the fence if you will. 

They purified chlorophyll themselves. It was free of iron, and when they added iron back, it protected as well. But it needed the iron plus the chlorophyll. So they still were worried thinking, well, maybe it's organic under those conditions. So that's 1925. Okay, so this is in rabbits. Inorganic iron won't correct. Alcoholic extracts or fresh cabbage, cornmeal plus iron will cure it, and chlorophyll corrected it as well. So we still haven't ruled out this potential vitamin as being necessary in these studies. So this was paper number one in this series. 

Now, I digress. You were all familiar with Steenbock. He's quite famous and started his patents then led to WARF which had a tremendous impact here at the university. He began then working following McCollum. McCollum left in a huff in 1917, and went to Johns Hopkins. So Steenbock carried on that tradition. And this just simply shows in yellow his report then of a stock ration for keeping rats that could then be used for the diet. And if you look through, here he's got the standard corn and casein and alfalfa, but they were also fed with fresh whole milk from the biochem barn just down at the end of Linden Drive. And they were able to sustain and use those animals. So there's the diet. 

One of the necessary components in biological analysis of foods. The next thing is an anemic rat model. In 1928, Waddell, Steenbock, Elvehjem, and Hart. So this is James Waddell, who's sort of the junior partner, Steenbock, Elvehjem, and Hart published series five. I've skipped the intervening four. Then published about using rats rather than rabbits. And what they found then, and I've sort of characterized it here. Here you can see a pink rat holding a penny, and an anemic rat, pretend anemic rat holding a dime or something like that. So they had a model, the anemic rat model, and they also had gotten to the point now where they could assess anemia rather exactly by just making a cut on the tail and directly measuring the hemoglobin levels. And I've taken, not this data which just shows a whole series of mice that all had low levels of red blood cells, but you can see over time, from zero to six or seven weeks, there was a progressive decline then in the hemoglobin levels. 

So they had a remarkably exact biomarker that they could use along with the diet and along with this anemia model. All three of those are necessary in this biological method of analysis. So you can study and see what the missing factors are that might promote blood building. And these animals, again, are all fed whole cow's milk, fresh daily, every morning. I should tell you that other researchers at the time couldn't develop anemia in their rats. They, too, were feeding milk, and they couldn't get anemia under those conditions. So there was something different going on at Wisconsin, and I'll touch on that in a little bit. So, James Waddell, he went off to work at DuPont after getting his PhD from Hart, and he said, "our weanling rats, restricted to a milk diet, developed a profound anemia in a few weeks and proved to be highly sensitive in their response to potent supplements." Now, this guy, in his review, complained about having to feed rabbits. Remember McCollum who was complaining about having to feed cows? He didn't like that they were variable and they ate a lot and so on and so forth. So he really liked this well defined rat model for this work. 

I should go back for a second and just point out that this publication number five was submitted on March 17, 1928. He reveled in this 1954 perspective that he really, or this retrospective, that he really started this work in 1926. Those Wisconsin biochemists were pretty clever in how they published as well. They didn't let the secrets out too fast until they really nailed something down. Okay, so remember the chlorophyll now? Is there protective factor, organic factor then, that's necessary? So, paper number six now in the series, again from the same cast of characters, started to look at iron salts and iron containing ash extracts. Again, the ash will get rid of the organic material. And let's see if there's something there. Again, this paper was also sent in the same time on March 17, 1928. Okay? And I'm not going to go through all the data, but here's one set of data here. Again, we've got these rats, okay, and if they're just given inorganic, so they're fed for about six weeks here, and their weight plateaus off and their hemoglobin levels are dropping if they're supplemented daily in this milk with .15 milligrams of iron, inorganic iron, or with 1 milligram. 

You can see there really wasn't much of an effect. But if they put in the ash of lettuce in two experiments, here you can see they get up to a plateau, at this point then they start supplementing with this ash of lettuce, remember we don't think lettuce has got anything in it, no calories, no food, no nothing, maybe taste if you put some nice vinaigrette on or something like that, but here there's something in that lettuce that allows those animals to quickly assimilate that inorganic iron. And you've got two examples here. So this ashing clearly said this protective factor is an inorganic, it's not an organic factor, it can't be a vitamin. They wondered, well maybe it was the form of iron. So these scientists very carefully took iron wire and dissolved it and made various salts. So they made iron sulfate, and you can see nothing happened. They made iron acetate, nothing happened. Citrate, phosphate, chloride. So no form, no iron salt. 

So that wasn't the clue either. There was something else, something beyond iron that was protective. Remember the single grain experiment? Now, Hart was a great one for this. My father was actually a graduate student, got his PhD from Elvehjem, and he was sent over by Hart, over across old University Avenue there used to be a grocery store to get some tomatoes to come over and feed some animals. So he liked testing various food stuffs themselves. We, faculty at the University of Wisconsin ought to remember, there was this stronger connection with what really went on out in the real world in those days. So they decided to test in this milk model five grams of dried beef liver, kidney, muscle, corn, or wheat, add that to the milk diet and see what happened. So the animals would become anemic on just milk, and they if they gave liver, five grams of dried liver, they actually started growing and their blood was improved, but that didn't happen with kidney, didn't happen with corn, didn't happen with wheat, didn't happen with muscle. So, clearly, the nature of the food, back to the proximate analysis idea, it makes a difference. There's something in liver that made a difference. 

Now, these had various amounts of iron, so they also fed the substances just providing then .15 milligrams of iron a day, and there, at that point, liver didn't do anything as well as the others. So it wasn't the iron, there was something else in the liver then that made the difference. So in 1928 then, ashed lettuce was just as good as lettuce in reversing the anemia. It's inorganic. Inorganic salts of a variety of types didn't work. Dried variety of foods, kidney, muscle, corn, wheat, they didn't correct it. A lot of liver would correct it, but if you just balanced it for iron, it wouldn't. So they concluded these ashes and extracts, in addition to iron, contained other inorganic substances vitally concerned with the building of hemoglobin. So, paper number seven then in this series, okay, remember this goes on to 10 or 12 but we're going to stop with this series at seven, is really the seminal paper in this study then. Now, note the publication just on the side. 

This is just 11 days later. So all the work in this paper wasn't done in 11 days. This is sort of like the bible. You know what I mean? They're skipping and telling the story a little differently here. But nonetheless, 85 years ago, and I thought I'd just point out then the cast of characters. So Hart came here, as I said, in 1906 just as Russell. Russell was famous for developing a TB test to take care of the dairy farmers because TB is rampant in milk. By the way, we ought to have somebody come and talk about raw milk versus pasteurized milk and tuberculosis and some other things to this audience. That might be a useful thing for some legislators to hear. Oh, shouldn't say that, should I? [LAUGHTER] So Hart then, Hart didn't have a PhD. 

Okay? He was Professor Hart. But Steenbock then got his degree in 1916. Waddell, James Waddell who went on to DuPont in 1926. Conrad Elvehjem, Elvehjem Arts Center, Conrad was very famous then for discovering what would cure pellagra. What animal do you think he used to cure pellagra? Do you think he used cows? People? Rabbits? Pigeons? He used dogs. Black tongue in dogs was the pellagra of dogs, and that was his animal model and that literally eliminated pellagra and led to the discovery then of niacin. Very, very key discovery as well. Not using an agricultural animal, I want to point out. Okay. And Todd is a player then that we'll get to later on when we switch over to zinc. 

So there was a close connection at that time with the faculty here at various drug companies, pharmaceutical companies like the ones who were trying to do something other than sell snake oil. So, as he said in that paper in 1928, we were fortunate to obtain for experimental purposes a generous quantity of a liver preparation from Eli Lilly which has been found to be very effective in the treatment of pernicious anemia and it was low in iron. Now, remember, in the '20s and '30s, pernicious anemia was like AIDS today. Not today, a decade or so ago. If you got pernicious anemia, you were going to die. You were going to slowly dwindle off for a couple of years and die, and it just wasn't curable. That's why people used to eat liver every single week, and I'm sure some of you remember those days. My father liked to talk about it and claimed he liked liver. At any rate, they began studying this. Again, they were trying to put to death this organic thing. 

So they would put in this milk diet, so here again are our rats and they grow for about six weeks and sort of stop going, and at that point they put in .3 grams of this liver preparation. And you can see alone growth was restored. So there was something in there that restored growth. So there are other substances, but the hemoglobin levels really didn't rise. But if they put in the liver preparation and also some iron as well, you can see there was an instantaneous increase with that preparation. So there was something in there that was very rich in that product, and the hemoglobin levels then are up at normal really within two weeks. So there was something remarkable there that allowed the use of iron under these conditions. You see how this biological method of analysis works? You have to have deficient animals, excuse me, a deficient diet, you've got to have animals that become deficient, and you've got to have a really acute biomarker, in this case that quick hemoglobin assay, so you can really detect when you've got something. Those were the things that were so central and the people where at Wisconsin did such a great job of assembling those mixtures to answer questions. And you can see they're bouncing around. 

They've got an idea here, but then they're off trying corn and wheat and other things as well and chlorophyll, Eli Lilly for fractions. So they've got something there. Well, what's the next thing you want to do? So let's ash that preparation and see if whatever is in the Eli Lilly preparation is there. So here you can see the ash of the Eli Lilly preparation and you can see, again, the minute they put that in, the hemoglobin, now they're getting to the point where they're actually going to show us four rats in one panel. So they did a hydrochloric acid digest. So this will redissolve some of the inorganic elements. Acid will bring those elements back up, and that was just as good. So this, whatever substance was, it was in the ash, it wasn't organic, it could be redissolved in HCL. They also did an HCL extract of a large quantity of that and used that as well and fed that here. Now, what they were really doing is every single day, a graduate student, James Waddell, would have ashed a certain amount of this Eli Lilly product in a crucible, and then he would pour in 2 CCs or how many ever CCs of milk the animal was supposed to get, and then grind it with the mortar. 

Okay, so there's a lot of work going on here. This isn't just so simple. So there they made one prep and just put the HCL prep in. Okay, that did the same thing. They also tried what was left after they dissolved the residue, and here you can see the hemoglobin levels didn't budge at all, and the animals, if anything, were losing weight. So it wasn't in the residual. It was something that was in the ash, you could extract with hydrochloric acid, and it wasn't in the residue. The last thing I'll show you here, how many of you took qualitative analysis lab back in chemistry? Remember that? The first day of that was a smelly day. What did you do? Okay, it smelled like rotten eggs. So you bubbled hydrogen sulfide through your solution in a precipitated cadmium sulfide, silver sulfide, and copper sulfide. 

So they made the hydrogen sulfide fraction of that HCL extract plus iron, and that also, very nicely, you can see here's animals not doing well, they put that in and their blood hemoglobin levels go up really well. These guys are clever, though. So they also swung for the fence on this one as well in the same paper. So the first trial of copper was done in a rat. And so Hart et al say this, "We made this trial of a copper salt because it fitted into our scheme of testing all inorganic elements as supplements to iron, which of these inorganic elements have been recognized as being present in biological materials." So they're going to go to the chemical shelf and pick out all the salts of metals that they've seen, inorganic substances they've seen and just test them one at a time. Now, today, we've have to have eight animals in a group and do statistics. They just did it one animal in a group or they used a litter and gave each littermate something different to see what would happen. And so they did this scheme. And the first rat they tried, rat 621, and there he is, okay? You can see he's getting milk for eight weeks. He grows for a while, and the last four weeks he doesn't grow at all. 

And, as they say, his hemoglobin levels then dropped from 12 down to 2.5. They're down right there, and they started putting copper in, .25 milligrams of copper, plus the iron, 5.5 milligrams of iron, and look at the hemoglobin. It just comes right back up. Look at him grow. So copper appeared to be that missing substance. So without the copper addition, the rise in the hemoglobin would not have occurred. So this was the discovery then, that this inability to assimilate inorganic iron was due to a copper deficiency, not due to an iron deficiency, using the biological method of analysis. So, as they said, "We think that this is the first experiment in the literature giving to copper an association with iron the specific function of hemoglobin building in a mammal on an otherwise satisfactory diet." Pretty nice, tight statement. You don't see those kinds of statements all the time in the literature. That's 85 years ago then under those conditions. 

So a liver preparation from Eli Lilly, the hydrochloric acid digest, the hydrogen sulfide precipitate along with supplemental iron, they all would reverse the anemia. Inorganic copper plus iron worked very effectively. So they said, "We have secured no evidence for the existence of an organic factor that's a vitamin necessary for hemoglobin synthesis in rats kept on a whole milk diet supplemented with .5 milligrams of iron." Now what's going on here? Why could they get this to work and somebody else? So the biochem dairy barn just down the way fed them fresh milk daily. It wasn't processed. It didn't go through a lot of copper pipes or anything like that. Okay? So they were getting copper deficient milk in that process, and I'm sure they weren't milking into a galvanized bucket under these conditions. That's certain as well. They also so on to say we might advance the orthodox, I'm not sure this was so orthodox, but it was suggested that the copper acts as a catalyzer for some reactions concerning hemoglobin building. So these scientists peering into this black box using the animal as their experiment model were really able to predict things way beyond their ken in terms of understanding what was going on with regard to nutrition, biochemistry, and metabolism. So, in the intervening little period here, manganese essentiality was demonstrated here, in 1931, Kemner, Elvehjem, and Hart. 

And magnesium essentiality in mice was also, here's mice, another animal demonstrated in 1932. This is McCollum working at Johns Hopkins now. So what I want to shift gears now and talk about sort of the last of this little story. And that's a paper that I used to think was published in 1934, but as I was preparing this and having to go back and find these little titles, I discovered that this volume, 107, was actually the first issue was actually published in December of 1933, not in 1934. And so this was received for publication in '33. There's E.B. Hart. "The purpose of this research was a further attempt to determine whether or not zinc does exert a beneficial role in the nutrition of the rat." So they've shifted gears, at least Hart has here, to see if we can find how many of these inorganic elements are really required in biology using this biological method of analysis. So this is going to take a series of things to do this well. And so Todd, again, this is Wilbur Todd and I know nothing else about him, but he published a paper in 1932 because they needed to develop a method for detailed, accurate biological zinc analysis of biological materials. And I've just outlined here their method that they reported then in the journal biological chemistry in 1932. 

So, notice the similarity here. So they're going to ash the sample, make the ash. So you've got inorganic. They're going to dissolve it in hydrochloric acid. They're going to use hydrogen sulfide precipitate it. They're going to reoxidize it with hydrogen peroxide. They're going to play around with the PH and throw something else, citrate, in that might key weight whatever else is in there, and then they're going to use hydrogen sulfide precipitation to get rid of the copper again. What's left is the zinc then. So can precipitate that phosphate. They wash it with alcohol and get rid of some other stuff. 

They dissolve it in strong acid, throw in some ammonium molybdate, which makes sort of a blue colored complex, and then they can analyze that for zinc. So they could screen different diet components and treatments to make a zinc deficient diet. So they set out, in this case now, this wasn't a mystery, they set out to say can we determine whether or not zinc is an essential nutrient for mammals. So they had to have a method of analysis. They also then had souped up their rat diet by 1933, and I summarize here in a little bit of detail, but "a complete ration for the rat should contain as an ample supply of carbohydrate, protein, fats," there's the proximate analysis. But they now knew we needed fatty acids, vitamins, minerals, and water. And they also noted that distilled water from the tap wasn't good enough. Excuse me, they really needed to redistill this water in order to make it free enough of metals that they could conduct these experiments. Again, a lot of careful attention to detail under these conditions. So here's their diet. 

This is virtually the same diet that I use. So they're going to use casein and egg white to provide the protein, but notice they took the milk, they precipitate it with acid and wash it, get rid of all the metals, and egg white was boiled, dried, and so it's about 19% high quality protein. This is exactly what we feed to rats now. Starch, again, this is hydrochloric acid treated so they get rid of all the metals and low molecular weight factors as well. They had a salt mixture, and I've got this down here. When I first started in grad school, people still talked about these salt mixtures. So the first one, here's the magnesium, that was discovered as essential in 1932, sodium chloride, potassium chloride, calcium phosphate. And then another salt mixture that's got the micro trace elements: iron sulfate, copper sulfate, manganese sulfate, and iodide. All present then. So they're putting all the minerals that they know about into this diet. 

They put in the fats and the vitamins. And so they made B12 from egg white. They sort of extracted rice polishings, just the polishings to get the anti-beriberi factor B1. It's sort of a dark syrup. They used fish oil to provide A and D, and they put that in corn oil and then they actually gave, orally then, some started off with one I noticed in the publication and some experiments up to three to five drops of this in the mouth of the rat every single day to provide the vitamins and the essential fatty acids then. And they still were getting too mils of fresh milk daily. We're the dairy state still. So this only contained around 1.6 parts per million zinc under these conditions. So, jumping right to, this is one of six experiments that they showed. We've got littermates that were fed this zinc deficient diet, and then they're staggered here by six weeks. 

But they all are growing like this, and you can see after, three sixes are 18, so after about 18 weeks this is the growth then of these littermates that were fed the zinc deficient diet, and if they were supplemented with 50 parts per million, you can see they grew a lot better. So they had a series of experiments in their actual paper that said if you put zinc in, they grew much better. And, in addition, these mice, after extensive periods of time, lost hair. So this is the ventral, the belly surface of this rat, and you can't really see it too clearly but you can see there isn't much hair there. So they lost hair as well. And then they did the experiment of repleting animals like this, and hair started growing back after five weeks with a supplement in a deficient animal of zinc. By seven weeks, it's gone. Head & Shoulders has got buckets of zinc. And that probably arises in part, I think it's also an anti-dandruff agent as well. So they showed if they repleted, they could correct that deficiency as well. 

So, rats were fed this zinc deficient ration. They had inferior growth. They didn't reach the same levels of weight. 50 parts per million of zinc then prevented the effect. Rats fed on the deficient diet lost hair, and if they were re-supplemented, the hair came back. So here's what they said. It seems evident that the ration was not entirely complete even though all of the known essential nutritional factors were supplied, that an additional factor or factors do exist and the water soluble B complex is rather definite, although their nature has not been elucidated. And so this Wisconsin team went on then to unravel the B complex. So 1937, that's when Elvehjem identified niacin as protecting against pellagra. Snell, working in bacteria, identified pantothenate as an essential vitamin as well, an organic substance that we can't make and so on. 

This wasn't done in dairy cattle. This wasn't done in pigs. This was done in experimental animal models. They also said another possibility was a lack of further inorganic elements, such as aluminum, boron, arsenic, and others. So there could be inorganic elements as well. So, from 1928 to 1933, really December of that year, five years, we saw a sweep of a whole series of new essential elements being demonstrated truly as being essential. All done here at the University of Wisconsin. So here's the list that you saw before. I've got the seven at the top, and then you can see copper in '28, manganese and magnesium '31 and '32, zinc in 1933, working in sheep then. Again, sheep are ruminants so they need inorganic cobalt and the microorganisms make vitamin B12. 

That was shown to be essential. Fluoride for dental caries was shown in 1938. And sulfite oxidase was actually identified as a molybdenum dependent enzyme in 1953 which kind of showed that molybdenum was also an essential nutrient even though you can't make animals deficient in molybdenum just by normal means. And finally, Schwartz and Faltz in 1957 demonstrated that selenium was an essential element. But there's arsenic that was in their list, boron in their list, and a series of others for which we do not have good proof that they actually are essential. So most of that heavy work was just done in about five years here at the University of Wisconsin and a few other places using this method of analysis that was developed in identifying the fat soluble and the water soluble vitamins. So I want to finish up now, we've got about 10 minutes to go, and I thought be might tie some strings around this just a little bit. So what in the world is that copper doing anyway? Well, we now happen to know about that, and it turns out that copper actually is necessary for a multicopper, that's a pretty good clue, ferroxidase. So this is an enzyme that oxidizes iron. Okay? Okay, excuse me, reduces iron. 

And that's actually attached directly to a transporter then that transports ferrous iron into cells. And so if an animal doesn't have enough copper for this enzyme, it can't put it in the right oxidation state and transport it into a cell. So that's why, even though these rats or rabbits were getting lots of inorganic iron, they weren't able to assimilate it because they couldn't get it inside their bodies. Now, how do you think that work was done? How do you think that discovery was made? That discovery was actually worked out in yeast by David Eide first, who's now a professor in nutritional sciences here, who showed that this multicopper oxidase is actually necessary then for simulation of iron in yeast and that explained that phenomenon. Now, what about copper? It's very difficult to make people truly deficient in copper, and there's been a lot of arguments about this. But there are genetic deficiencies in various enzymes and other things like this. One of these is a copper transporter, ATP7A, that's involved in Menkes disease. So these individuals have a defect that doesn't allow them to transport copper into the body at normal rates. It's X linked. Okay? So even though there there isn't a deficient diet, they just can't get enough copper into their bodies and into cells as well to do this. 

And so this is characterized by growth failure and then a deterioration of the nerves, the nervous system, mental retardation. The kids usually die by age two. This little diagram just shows this transporter that allows copper to go into cells, and there are some sites along here that help guide the copper perhaps into that transporter. So that biological method of analysis simply was uncovering what we now can look at at a molecular level. What about zinc? Well, it turns out that there are proteins have sulfur containing amino acids cysteine and histidine, and they actually then will key weight zinc. And they make something that's called a zinc finger, and that's what's diagrammed here. And you can have proteins that have up to 37 of these zinc fingers. These zinc fingers can be spaced just right, and with the right amino acids they can actually scan the way a pianist goes along the piano keys and intercalate right into the DNA. And so transcription factors, factors that control gene expression, have zinc fingers on them, and the zinc is necessary for those fingers then to find the right place and turn on and turn off genes in our system. That might be related to growth, wouldn't it? So, again, that role has now been moved forward. 

And this is just an example of this. So, first we'll talk about, I  think I swiped this diagram years ago from Wes Pike, one of his publications, but these are a whole series of steroid receptors. These are proteins that recognize thyroxine or recognize vitamin D. Thyroid hormone, estrogen receptors, androgen receptors, progesterone receptors. And they all have a zinc finger region that guides them to just the right place in the DNA to turn on and turn off genes. Okay? So there used to be kids in Egypt and in the near east, this is an Iranian boy, 16 years old, he only is about 54 inches tall, only weighs about 66, he's 16 years old, no pubic hair, hasn't really sexually developed. This is due to zinc deficiency. And a guy by the name of Persad discovered this in the '60s actually. So that's nutritional zinc deficiency. Again, an impairment of growth. 

And you can imagine if all these growth factors aren't working because there isn't enough zinc, you're going to be in trouble. In addition, there are zinc transporters, and so there are genetic diseases. This happens to be a disease called acrodermatitis enteropathica, and this is due to loss of one particular zinc influx transporter. It happens to be Zip4. And, again, these kids develop a rash, they don't have the skin around the various openings, and so on and so forth. They can be treated with high levels of zinc. It's interesting, zinc and copper move into the body in various places in parallel, so they have to give them extra copper as well in order to balance that out as well. So there are genetic and nutritional zinc deficiencies that are possible. Okay, so I did better than I thought I was going to be. So I want you to think about this now, so when I showed you that list, we're going along, five elements or so in five years, the biological method of analysis is just working like hell, and then all of the sudden what's happened since then? Okay? And the same thing is largely true in the vitamin field. 

 Okay? And yet, if you go to the health food store or you go to Stop & Go, there's somebody hawking some pills of something or other that isn't one of the known vitamins, and telling you that might prevent a cold or telling you that might do something for you. But we haven't been able to demonstrate that those really are essential. So, why is that? Why has this stalled?  Have some ideas? I'll maybe have some discussion at the end of this. But let me postulate then, I don't think it's just because the state has stopped supporting the Ag School here at UW Madison so well. Though, that might be a little to it. But I think I sat down, I haven't thought about this a long time because I don't give this kind of lecture, but let's think about some things. Maybe people can't make diets that are sufficiently deficient in some of these other nutrients. A boron deficient diet or a tungsten deficient diet or something like that. A graduate of UW Madison, Frosty Nielsen up in Grand Forks, struggled with this. He used plastic cages and double distilled and triple distilled and everything else and really failed to really demonstrate nickel essentiality or something like that. 

So that doesn't seem to be the only reason. Maybe something called homeostasis is involved. Maybe we're so well-tuned. An ape out in the woods doesn't read that pomegranate and decide that it's got the right nutrients and then decide he's going to eat it and then go over here. Number one, he just eats because he's hungry and hopes that the good stuff comes along with the bulk. But maybe homeostasis is there to really protect our supply of nutrients, so we really hang onto it if it's in short supply, and maybe we get rid of it if it's an oversupply. So homeostasis, homeostatic mechanisms may be one of the reasons why we can't do that. Another highly possible thing is that there are a series of overlapping protective roles these minerals, these vitamins play, essential roles, and so there may be several dehydrogenases or reductases that do basically the same thing, can work on the same substrates, and if one of them is low, another one goes up a little bit and just cuts around it. That's likely to be something that's going on. I've got a situation like that in my research going on right now. 

It's possible that there are minor pathways, okay, that might provide some synthesis. We now have a requirement for kaolin. Basically that's ethanol with a bunch of amino groups and methyl groups on it. And it's been discovered that we just don't make enough of it, humans don't make enough of it now to fully supply those needs. There may be some genetic differences in us that affect that. We've known rats grow so fast that we've got to kaolin in the diet. Again, maybe if we found the right animal model, we might do that. So there may just, we might not have some good models for lutein deficiency or cryptoxanthin deficiency or boron deficiency, something like that. So there may not be good models. There's also the possibility that we only really require that nutrient at one particular place. 

Citrus have to get buckets of boron. So, grapes, oranges, they use lots of borax/boron. But almost all plants use that in reproduction. And it turns out that if you make a plant deficient in a gene that's involved in the elongation of the pollen, the pistil, inside the pistil, that stops, and boron is necessary for the cross-linking. So boron may be actually essential in reproduction. So there's only one phase that all plants need. Just a little bit of boron, and there's so much around they don't need it. Or maybe that you only need it under rare conditions when there's another enzyme that's been knocked out or you're under stress or you're fighting off a cold or something like that. So there are a whole series of reasons why we haven't found that, but I really don't know. So I'd sort of like to finish just a little bit. 

What I wanted to do is get you back into the lab and see some of the basic science on how it was reported, and I think I've done that. I think I wanted to emphasize how important this biological method of analysis was. That replaced, clearly, the chemical method of analysis, and I would propose that perhaps a genetic method of analysis in combination with some of these are likely to solve some of these things. So I told you about molybdenum. So it turns out that there's an enzyme sulfite oxidase that requires the molybdenum cofactor. We can make it, but if you don't have that enzyme, at about age 20 people suffer mental retardation and die. There's another enzyme where the same thing happens, and there's several enzymes that are involved in the cofactor. They're all there. So even though we can't make animals or people molybdenum deficient, we clearly know we need molybdenum because we need that enzyme, we need that cofactor. So it's clear that it's essential, and things like that may be done to solve this. 

So I hope I emphasized in that the role of a good animal model, a good diet, a good biomarker, and a bunch of stuff to test, whether it's tomatoes from across the street or butterfat or milk or whatever as well, and emphasized multiple models. I want to repeat. We talked about cows, we talked about rats, we talked about pigeons, we talked about mice, we talked about rabbits, we talked about rats. There's only one of them that's an agricultural species, isn't it? And that's what put Madison on the map. Please remember that. And these things were used to solve economic problems, help the dairy industry, cure pellagra, a problem not so much in Wisconsin but certainly in southeast America pellagra was a huge problem for people who ate largely corn and things like that. And so these solved state of Wisconsin, world economic issues as well, and I think that's a challenge that the state needs to continue. We need to address some of those issues rather than nice issues a little bit more so we maintain credibility with the taxpayers. Madison, the vitamin A researchers, their grad students, they had a profound impact then on nutrition and biochemistry at UW Madison and the nation and the world. And that all stands on the shoulders of those few people in the teens, in the nineteen-teens and the '20s and the '30s that did this outstanding work. 

And I, continuing in the mineral area, owe them a debt of gratitude as well. So I've loved telling you, in detail I guess, a little bit of a story, but I hope you found it interesting and I'd love to answer some questions. Thank you. [APPLAUSE]