cc  >> Cathy Vrentas: Hi, everyone,  welcome to Wednesday Night  at the Lab here at  the Biotechnology Center.  Tonight we're welcoming Denise  Ney who is a professor in the  Department of Nutritional  Sciences.  She got her PhD in nutrition  from Cal Davis,  and she's a Billings Bascom  Professor and a Waisman Center  investigator here on campus.  She's had a variety of roles  including department chair,  director of programs including a  campus training program in  nutrition and a program in  dietetics.  And her work, which she'll be  talking about today, looks at,  one of the angles is looking at  the nutritional management of  the disease Phenylketonuria.  And she works with the  Biochemical Genetics Clinic at  the Waisman Center on this  project.  So please join me in welcoming  her to Wednesday Night at the  Lab today.  (applause)  >> Denise Ney: Hello, everyone,  and thanks for coming out.  I'm competing with Concerts on  the Square, I hear.  So I'm very pleased to have this  opportunity to tell you about  our research which shows that  glycomacropeptide improves the  diet required for  Phenylketonuria. 

 PKU is a disease which has  probably touched all of your  lives in here if you may or may  not know that.  And the reason for that is that  since about 1965 every baby born  in the United States is screened  for PKU before they leave the  hospital.  And I'll tell you more about  that.  So in a nutshell, what's the  whole story about what have we  discovered.  We have identified a protein in  cheese whey, GMP we'll call it,  that can be used to make foods  and beverages for individuals  with the rare genetic disease  PKU.  And this disease, PKU, requires  a life long low phenylalanine  diet to prevent brain damage.  This research, I think, is  really exemplifies the Wisconsin  Idea in action.  That is the boundaries of the  university are the boundaries of  the world.  Because this is an international  problem, the diet for PKU.  And I've had the opportunity to  speak all over the world about  our research. 

 And there's great excitement for  the potential to improve this  diet which is the mainstay of  treatment of PKU.  One of the fun things for me is  this a very interdisciplinary  project.  I've been involved in this  project for six years and when I  think about it it's amazing what  we've accomplished with really  not that much money, and part of  this has been because of the  wonderful cooperation that we  have had across campus.  On the left you see Mark Etzel  professor of food science, who  actually started this whole  process about 10 years ago when  he identified GMP in cheese whey  using a small grant from the  dairy industry.  And he realized it had potential  for PKU, and he contacted the  dieticians at the Waisman  Center.  And you see myself and then you  see Sandy Van Calcar and Sally  Gleason who are the key senior  dieticians at the Waisman Center  and they agreed it had potential  and essentially Sally Gleason  recruited me into this project  about six years ago, and it was  a very good time for me because  I was stepping down as  department chair and wanted a  new project, and also I did my  master's degree working with  inborn airs of metabolism at the  University of California San  Diego so this is an area which  is kind of dear to my heart.  And I understood it and it was a  high risk project but I said  let's go for it.  Let's see what we can do.  And also is Erin MacLeod on the  right who is a PhD student  working on this project.  So we started out with a small  grant from the graduate school,  some -- funds because Mark had a  patent and it wasn't licensed. 

 I leveraged that into some money  from CALS to do some experiments  in PKU mice and I went to NIH  and I got the grant right off.  Which enabled us to do the small  clinical study which I'll tell  you about.  We have had support, of course,  from the Waisman Center which is  the Waisman Center in their  biochemical genetics clinic  cares for 164 individuals with  PKU.  And all of our subjects came  from the Waisman Center.  The School of Medicine, we  admitted subjects to the  hospital for an inpatient study.  The ICTR, the Institute for  Clinical and Translational  Research.  The Wisconsin Center for Dairy  Research developed all of our  GMP food products, and not shown  here is Kathy Nelson who did  this.  And also the PKU community.  I had had grants from MACPAD,  from the Mascho Foundation, the  Mascho Foundations are really  big leaders in PKU in the United  States.  They're a wealthy family whose  granddaughter has PKU, and they  were very instrumental in  offering support for this  project early on. 

 A little outline for today.  I know I have an audience with  lots of different background,  I'm sure, so I'll keep it  general and you can ask me  questions.  We'll start with PKU.  This rare disorder affects the  metabolism of the essential  amino acid phenylalanine or PHE  as I'll refer to it.  It's caused by a defect in the  liver enzyme, phenylalanine  hydroxylase, which converts  phenylalanine to tyrosine.  And when an individual is born  with PKU, immediately when they  get a normal diet, breast milk  and formula, they can't  metabolize the phenylalanine and  it accumulates in their blood to  very high concentrations, maybe  20 times normal.  High phenylalanine in the blood  is transferred to the brain  where it is toxic.  And if a baby is born with PKU  and is not put on the treatment,  which is a low phenylalanine  diet, within a few weeks of life  they will suffer severe and  permanent mental retardation.  The incidence is about one in  10,000 births.  We have four to five babies who  are born every year in Wisconsin  with PKU. 

 There's about 15,000 treated  cases in the United States and  about 50,000 to 70,000 cases  worldwide.  The disease was first described  by a physician, Dr. Folling, in  Norway in 1934.  And I think I'd like to tell you  the story on how this was  figured out because I think it's  a very interesting story.  Basically there was a family who  had two children who were  mentally retarded.  And they actually lived in Oslo.  They were fairly well-to-do.  The father was a dentist and he  had asthma.  And these children who are  retarded have, there's a very  strong odor associated with the  phenylketones that are excreted  in the urine and the phenyl  acetate in particular.  And this odor of the children  aggravated the asthma of the  husband.  And the mother would not give  up. 

 And they went to the finest  clinics in Europe.  At that time they went to  Heidelberg, they went to St.  Petersburg, they went to Paris  and no one could help them.  And the mother very simply  explained, and this was her  proposition, she said couldn't  this odor, especially in the  urine, couldn't this have  something to do with why my  children are retarded.  And then she went back home and  she found out about a physician  in Oslo, right in their home  town.  And this physician, he liked to  work in the lab.  He had a strong background in  biochemistry.  He didn't see many patients but  he agreed to see the family.  And she said can't this odor  have something to do with my  children.  And he said, well, it's a  possibility. 

 He said, bring me in their  urine, bring me in liters of  their urine.  And she did.  And he did an old fashioned  organic chemistry and he  isolated and he found that  phenylpyruvic acid was very,  very high in the urine.  And he surmised that this must  be due to a metabolite of  phenylalanine.  Of course they couldn't measure  phenylalanine in blood at that  time, but that's actually how it  was diagnosed.  Fortunately, mental retardation  due to PKU because of newborn  screening is very rare in the  developed world.  The newborn screening was first  initiated by Dr. Guthrie in  1961.  There is a very nice link to  this campus and Harry Waisman,  for whom the Waisman Center is  named.  In that Harry Waisman was really  a key figure in lobbying at the  national level to require  legislation in the United States  that every baby born in the  United States be screened for  this disease.  And this was the first disease  that screening was used for in  this country, I believe. 

 Now we, the Wisconsin State Lab  of Hygiene screens with tandem  mass spectrometry.  They screen for 48 disorders.  Wisconsin is in the lead with  newborn screening nationwide.  We have a very, very good system  for this in our state.  The treatment.  The treatment for PKU is  lifelong adherence to a low  phenylalanine diet.  This diet was actually first  developed in 1951 by Bickel in  Heidelberg.  And an individual with classical  PKU may have their diet  restricted to 200 to 500  milligrams of phenylalanine a  day.  Protein contains between 5% and  10% phenylalanine, so all sorts  of protein in our diet.  So this amounts to a restriction  to three to six grams of intact  protein a day. 

 This is nothing.  Most of us in the room eat  100-grams a day as Americans.  The diet includes two features.  The first is that natural foods,  all of which contain protein,  even fruits and vegetables, are  severely restricted in the diet.  We're talking about weighing  broccoli or peaches or bananas  or things like that, to not have  too much of these foods for the  phenylalanine content.  And even the breads that we eat  and our pastas are too high in  protein and that's phenylalanine  so we have to have special low  protein pasta and bread  products.  So we severely restrict the  natural foods to the point that  the diet would be inadequate.  So we have to make up for it.  And we do that by using an amino  acid based formula that is  phenylalanine free.  And this provides the majority  of protein, energy and  micronutrients. 

 It's fortified with vitamins and  minerals, and most people with  PKU consume three to four cups  of this formula a day.  And that's the basis of their  diet.  This kind of gives you an idea  about the phenylalanine content  of food.  Remember that 200 to 500  milligrams a day for someone  with classical PKU.  So you can see all of these  foods are completely eliminated.  Even bread, 132 milligrams.  And now we get to the foods that  are allowed.  French fries which are a big  treat.  Lettuce, apples are fairly low,  and apple juice, in  phenylalanine.  If you drink diet soda you've  seen the can and the label on  the can for the sodas that are  sweetened with aspartame which  says "phenylketonurics avoid."  That's because, one can, 100  milligrams of phenylalanine. 

 Aspartame is a diet peptide of  aspartic acid in phenylalanine.  So you get the idea here.  So then we have the formulas  which are the mainstay of the  diet, and they smell bad and  they taste worse.  It's difficult.  They have improved over the  years.  Now this diet in infancy is not  a problem because the babies  will take the amino acid  formula, it's fine.  The problems start usually when  the children go to school.  They don't want to be different.  And they certainly don't want to  take that formula to school for  lunch.  For adults, over 50% of the  protein and the calories still  need to be provided by the amino  acid formula and other amino  acid based medical foods. 

 Without the formula the diet  cannot be adequate.  There's protein deficiency; it's  deficient in micronutrients.  In clinic we see individuals  with PKU who don't follow their  diet, who don't take their  formula, and many of them  frankly have very poor  nutritional status.  So the problem is that most  adolescents and adults with PKU  cannot follow the diet.  They can't drink the formula, as  the mainstay of their diet.  And this results in severe  health consequences.  It varies depending on the  individual.  Could be from as slight to  headaches, having difficulty  concentrating, thinking,  breaking out in a rash, being  very nervous, having tremors.  Children in school who are not  following their diet, it's very  clear.  They can't learn, they can't  concentrate. 

 Could be from that extreme,  could be to not following your  diet bringing about paralysis,  the most severe case.  Women who are pregnant with PKU,  their babies could have  congenital anomalies.  So there are some serious  consequences to this problem of  not being able to follow the  diet.  And this is a life long  treatment.  Okay, now we come to the cheese  part.  Cheese is a high protein food,  not for PKU.  But I'm sure most of you realize  that in Wisconsin about 95% of  the milk that we produce goes  into making cheese.  We don't have a big enough  population to drink all of it.  So we have, we're the cheese  state.  So when we make cheese, we start  out with milk and we add rennet  which contains enzymes which  coagulate the casing protein in  the milk to form the curds or  the cheese and this leaves whey. 

 Lots of whey.  In fact, for every 10-pounds of  cheese you get 90-pounds of  whey.  So we have a lot of this in  Wisconsin.  So it made sense that the dairy  industry gave Mark Etzel a small  grant and said look around and  see what you can find.  Maybe there's something useful.  And he did find something  useful.  So GMP is a natural whey protein  produced when making cheese.  It's contained, this is the  whey, it's contained in here.  And the thing that's special  about GMP is in its pure form it  has no phenylalanine.  It's the only natural dietary  protein that we know of that  doesn't have phenylalanine. 

 So thus it is uniquely suited to  being used in the diet, the  low-phe diet for PKU.  Now there is one little glitch  here and that in the process of  separating the GMP, which  constitutes about 25% of the  protein in the whey, the process  of separating it from the whey  it does get contaminated with   some of the other proteins in  whey that do have phenylalanine.  So it ends of having a trace of  phenylalanine.  Because it's simply too  expensive to get the pure GMP at  this point.  This shows the essential amino  acid profile of GMP in the  maroon compared to an average  dietary protein.  And there's two points I'd like  to make.  The first is that the GMP has a  very unusual amino acid profile.  We don't know why, it's how  nature evolved it, part of the  milk fat globule membrane.  But anyway, GMP is very high in  threonine and isoleucine.  And both of these amino acids  fortunately are nontoxic and  they're what we call large  neutral amino acids. 

 And this actually becomes very  important in terms of the  efficacy of GMP in the PKU diet  which I'll tell you more about.  The second point is that it's  not a complete protein and we  have to supplement with five  limiting amino acids that are  circled on the X-axis in order  to make it a complete protein.  But even when we do this we end  up with something that's 80%  intact protein and about 20%  supplemental amino acids to make  it complete.  So we have the Wisconsin Center  for Dairy Research, primarily  Kathy Nelson, has developed good  tasting, low phenylalanine foods  and beverages made with GMP as  shown here.  And we have done sensory studies  with individuals with PKU and  they're acceptable to  individuals with PKU.  I wouldn't say that they taste  excellent or great that you'd  want to go out yourself and  substitute them.  I would say they taste good, but  compared to the amino acid  formula they taste great.  And here we see Jessica  Zimmerman, and she has PKU and  has helped us quite a bit with  some of our publicity.  And on my website there is a  link to a show that  Frederica Freyberg hosted called  "In Wisconsin."  It's about a 10 minute show that  Frederica did on our research.  You want to hear more about  Jessica and her story please go  there and click on that link and  you'll have some background on  her and the whole project. 

 Which probably will be more  meaningful after this session  today.  So to summarize, the GMP  provides an alternative to amino  acid formulas which are  essential in the PKU diet to  provide for adequate protein and  energy and micronutrient intake.  The advantages they have is they  taste better, variety, you can  make foods from GMP.  From amino acids you're kind of  limited.  You can make drinks and you can  make gels and you can make salad  dressings and a few other things  like that but amino acids don't  have the properties to like make  into a cracker or cereal, etc.  And convenience, many of these  foods that we can make are  convenient and could be, for  example, taken to school like  the bar could be taken to school  in the lunch box in place of  amino acid formula.  So moving on to our research, we  started out with, again, money  from the graduate school and  from CALS with some studies in  PKU mice.  And this is a really neat model,  the PKU mouse model.  It was actually originally  developed here in McCardell labs  by Bill Duff.  And it's neat because these  animals have the same what we  call the phenotype as people  with PKU where it's not treated  with diet. 

 They have the same defect and  same enzyme that has been  knocked out.  So it's a very useful model for  us.  So the first thing we had to  know is could we make this  nutritionally adequate because  the plan was to feed this  protein as the primary source of  protein in the diet of someone  with PKU.  And these data showed that GMP  supplemented with those limiting  amino acids can provide a  complete source of protein that  supports growth in weanling  mice.  And you can see here that mice  fed casing compared to GMP for  six weeks, weanling mice who are  very, very sensitive to an  adequate diet, they show an  identical growth rate.  So yeah, we can make it  nutritionally adequate.  We know what to add to.  Now in the PKU mice as expected  we saw an increase in plasma  concentrations of these nontoxic  large neutral amino acids,  threonine and isoleucine, the  ones that are so high in this  protein, compared to an amino  acid controlled diet.  The mice showed that plasma  phenylalanine decreased 11% in  the PKU mice that were fed GMP  compared to the amino acid diet.  And again this is the same  phenylalanine intake. 

 And this comparison of diets is  pretty much what it is in the  human diet.  An amino acid based diet or in  this case the GMP diet.  Now this is interesting.  We found that brain  concentration of phenylalanine  was reduced 20% in five areas of  the brain in the PKU mice fed  GMP compared to amino acid diet.  And again it's the same  phenylalanine intake.  Now this is exciting because the  toxicity of the high blood  phenylalanine concentrations  occurs when the phenylalanine  gets in the brain.  That's where the damage occurs.  And the effect of this 11%  decrease in the plasma was  magnified in the brain for lower  brain concentration.  So how do we explain what's  happening?  So it comes back to the large  neutral amino acids of which  threonine and isoleucine are so  high are considered large  neutral amino acids.  So the way we explain it is that  we believe that GMP provides a  food source of large neutral  amino acids. 

 In particular, threonine and  isoleucine.  And what happens is there is a  transporter across the blood  brain barrier.  There's competition to that  transporter to transport amino  acids into the brain.  And the high serum  concentrations of threonine and  isoleucine compete with  phenylalanine, because they're  so high, for entry into the  brain.  So that reduces the amount of  phenylalanine that's going in.  It's a competition.  There's similar transporters and  similar competition in the  intestine, could explain why the  blood levels of PHE were lower  in the mice.  Now this idea that I'm  explaining with large neutral  amino acids in this competition  of the intestine and the brain  is well supported by evidence in  the PKU field.  In fact, when supplements of  large neutral amino acids are  given they have actually shown  based on magnetic resonance  imaging that brain  concentrations of phenylalanine  are reduced.  And one of the therapies for PKU  if you have difficulty following  your diet is to take supplements  of large neutral amino acids. 

 Now this involves eat your meal  and you take 20 or 30 pills with  the meal of large neutral amino  acids and competes with the  phenylalanine for absorption.  So again there's good evidence  for this hypothesis that the  large neutral amino acids in GMP  are competing with phenylalanine  for transport possibly across  the gut as well as into the  brain.  Now to go on to our clinical  studies in individuals with PKU  that were made possible for the  NIH grant that I secured.  So we did an inpatient metabolic  study.  We had 11 individuals with PKU  and they were between about 12  and 31 years of age.  This study had three stages to  it.  The first that we worked with  subjects for two to three months  really to get them in good  control and to get them, they  weren't taking their amino acid  formula in sufficient amounts  the dieticians worked with them  to make sure they were taking it  so they could be in our study.  So after that we knew how much  phenylalanine everyone could  tolerate and that's the level we  used for our inpatient study.  The subjects then followed their  usual amino acid diet for four  days at home and then they were  admitted to the Clinical and  Translational Research Center at  University Hospital for six  days.  The first two days they  continued their amino acid diet  and then we switched them over  to the GMP diet. 

 And the GMP diet replaced all of  the protein equivalents from the  amino acid formula with foods  made from GMP.  And the subjects were actually  on a one day cycle menu for this  whole period, we just switched  the amounts and you got GMP  pudding or amino acid formula  and they were very, very  cooperative.  We kept constant the amount, of  course, of phenylalanine intake,  the energy, the protein.  They were really in the hospital  to follow the diet.  It was the only way.  We had to feed them, it was such  a special diet.  At the end of this study there  were no negative health effects.  No changes in health status,  with the amino acid diet  compared to the GMP diet.  They were followed very  carefully, physician visits  everyday, examinations, blood  work.  And the GMP diet was well  accepted. 

 In fact 10 of the 11 subjects  said if it was available they  would eat the GMP instead of the  amino acid formula, they  preferred it.  Now this shows these plasma  large neutral amino acids, same  thing that we observed in the  mice.  That is, when you start the GMP  diet, these are the two days on  the amino acid diet, and then we  start the GMP diet.  We go for four days.  And you can see there's a  significant increase in both  isoleucine and threonine.  It reaches a plateau.  It doesn't keep going up.  But there is a significant  increase.  So this would be consistent with  the idea that these high plasma  concentrations of threonine and  isoleucine could compete with  phenylalanine to decrease  phenylalanine transport into the  brain.  We're following up on this by  doing some magnetic resonance  spectroscopies work at the  Waisman Center. 

 But we don't have any results  yet, but we do have scans on our  patients.  We did not see a significant  change in postprandial or after  breakfast plasma phenylalanine  concentration with GMP compared  to the amino acid diet.  You can see some went up, some  went down.  I'm not sure we excepted to see  a significant change, although  if it would have all gone up we  would have been worried, of  course.  But this was a very short period  of time.  This was really a safety in  efficacy study.  And then we asked the question,  somewhat indirectly but in a  meaningful way, we asked the  question does GMP improve  protein retention compared to an  amino acid diet.  And the reason for this is that  GMP is an intact protein with a  slower rate of digestion and  absorption compared to amino  acids.  And in fact, there is some very  good literature, recent studies  with stable isotopes showing  that intact protein is better at  promoting protein retention and  protein synthesis than amino  acids are, as shown in this  slide.  The basis for this idea really  that intact protein is better  utilized for protein synthesis  relates to the fact that intact  protein like GMP is slowly  digested and prolongs the plasma  amino acids concentrations  compared with free amino acids. 

 So here what you see is with  time the changes in the total  plasma amino acid concentration  on the Y-axis.  And the lower line here, this is  cottage cheese.  That's what, after you eat  cottage cheese what your plasma  amino acid profile looks like.  And see how it's delayed and  it's blunted compared to these  other peaks.  These are the amino acids that  are in cottage cheese.  So they're disposed of very  quickly if it's in the form of  an amino acid.  And this is the time when we  sampled our subjects in the  hospital.  So our data support this notion  that intact protein from GMP  improves protein retention  compared with an amino acid  diet.  Because when you look out here  on the last couple days of the  GMP diet, these are the amino  acid concentrations in blood.  They're significantly higher  than they are on the amino acid  diet. 

 And probably most important is  that the BUN is significantly  lower at this time point  compared to, that's the blood  urea nitrogen, compared to the  amino acid diet.  And when your body makes urea  out of your amino acids in your  body that means it's degraded,  it's no longer available.  So there's less degradation of  amino acids with this GMP diet.  And also supporting this we have  higher postprandial insulin  levels with GMP compared to  amino acids.  And insulin is known to inhibit  protein degradation and  stimulate protein synthesis.  So to conclude, our out-patient  case study.  Now this is one adult male  subject.  Matt is his name.  And if you were to watch the  video he is also interviewed and  his story is told in that video.  And I think it's a very poignant  story about the importance of  following your diet. 

 So Matt was in the inpatient  study but he agreed to be our  guinea pig for the out-patient  study, he liked the GMP.  And this study involved three  weeks at home of following his  usual amino acid diet.  And then he went on the GMP diet  for 10 weeks and then he went  back to the amino acid diet for  two weeks.  And during this period he had  weekly blood draws to check his  plasma amino acid, his PHE  levels.  His menus were planned for him.  We sent him a lot of food.  He did a lot of things, it was  in hunting season, he went  hunting and we sent food there  when he went hunting and all  kinds of different activities.  He was great to follow the diet.  And during this 10 weeks at home  the GMP foods replaced all of  his usual amino acid formula.  And we always have our subjects  pick which GMP foods they want  to use in their diet and Matt  picked just these three foods. 

 The GMP sports beverage, the  pudding and the snack bar.  So that's what he had instead of  an amino acid formula for 10  weeks in addition to his small  amount of natural foods.  The GMP sports beverage is worth  making a few comments about.  I think it's a very good  product.  It really sort of tastes like  Tang or Gatorade.  It's sort of a bland tasting  beverage and really very  acceptable.  And he liked it.  He liked it very much.  And one of the things that he  did with the GMP beverage is  that he liked it so much that he  spaced it over the whole day.  That he would, of course, have  some in the morning but then he  would save some in the afternoon  or in the evening. 

 Now with his amino acid formula  he didn't like it.  So he'd mix it up in the  morning, 24-28 ounces, he'd  drink it all at once in the  morning and get it over with.  That would mean he didn't have a  source of protein the rest of  the day essentially, other than  the small amount, may be 10  grams, provided by food.  So anyway Matt showed a, he  showed a significant drop in his  blood PHE levels, 13% to 14%  with two methods of analysis.  Full plasma amino acid analysis  or the -- spectroscopy for the  blood finger stick PHE  concentrations.  And we don't know why this  occurred by I think there are  two possibilities, and probably  the top one is that he spaced  the GMP protein throughout the  day.  And we know that makes a  difference to reduce blood  phenylalanine levels.  Because if you drink it all in  the morning, which a lot of  people do, or children will not  take it to school, so breakfast  and maybe evening, in between if  you don't have a source of  protein you're breaking down  your protein and you're  releasing PHE into the blood  from that protein breakdown.  So I think in this particular  case is a very good possibility.  Now the other possibility is  that this intake of the large  neutral amino acids in GMP  provided for decreased  absorption of phenylalanine from  the gut. 

 We certainly know that if you  take concentrated tablets it  does that.  We don't have any direct data to  support that hypothesis, but  it's certainly an option.  But what I think this shows, and  when I speak with PKU audiences  around the country and I talk  with the families and their  children and they go to school,  the lunch box is a real  challenge because usually a  student may start out taking a  thermos full of amino acid  formula to school but the first  time they get teased that's the  last time they will take it.  So it's hard to get a source of  low phenylalanine protein to  provide to the students when  they're at school, when they're  active, when they need  something.  And this is a plus for the GMP.  So in summary, our research to  date shows that GMP enhances  taste, variety, you can make  foods out of it, and  convenience, you can take a bar  for lunch or a pudding of this  diet.  That the GMP improves protein  retention by decreasing the rate  of absorption of amino acids and  it makes it easier to eat a  source of low-phenylalanine  protein across the day.  All of this is going to favor  utilization of phenylalanine for  protein synthesis and reduce  blood PHE levels.  And lastly GMP may decrease  phenylalanine levels in blood  and brain.  We need longer term studies and  we need more research in this  area. 

 And I'm very excited about the  possibility of conducting a  two-site clinical trial.  I submitted a large grant to the  Public Health Service and I have    a very good score.  And I think I will know in  September, but I think this  study will be funded.  And we are going to study 30  individuals with PKU over a  period, it's going to take about  four years to complete it.  Half of the individuals will be  from Madison and half of them  will be from a children's  hospital in Boston, associated  with Harvard university.  And we're going to do a  randomized controlled cross-over  study.  We will study 12 years through  young adulthood.  So the subjects will receive  both an amino acid or a GMP diet  living at home in a random  cross-over fashion.  We'll learn about acceptability,  what it does to their blood and  phenylalanine levels on a  long-term basis, and so we're  very excited about that  possibility and perhaps I'll  come back in a couple years and  tell you about it, for the  concluding chapter of it.  So thank you for your attention. 

 I would be happy to answer any  questions.  (applause)