Dr. Baggott

September 8, 2008

11:00-12:00

(Note: these all follow his lecture overheads. I was unable to follow them myself, and thus give them the appropriate overhead heading. If anyone who was able to piece it together (in terms of the name of the overhead that goes with each amount of material) would like to insert them, please feel free to edit the document and do so. I apologize that it lacks cohesion because I didn’t know what he was talking about when. Thanks for your patience!)

What happens when we block this with methyltrexate?

You interrupt the cycle or block the synthesis of deoxy-TMP. When we looked at 5-flourouracil,which was an irreversible inhibitor of deoxy-TMP, or thiamidalite synthase rather. In your practices you’ll come across many individuals who are treated with either 5-flourouracil, which is metabolized to 5-F-dUMP, or methyltrexate, which is a dihyrdofolate reductase inhibitor. These are the kinds of things on medical and dental boards. 5 F-dUMP or 5-flourouracil-- if you don’t want to metabolize it or they ask what the drug is--inhibits thiamidylate synthase and is an irreversible inhibitor and then methyltrexate is an inhibitor of dihydrofolate reductase.

Now let’s catabolize some purines.

We’re not going to do the catabolic pathway of pyrimidine (it’s not interesting) and there’s not that much salvage of pyrimidines but there’s salvage of purines. We’ll look at the catabolic pathway for purines, and some of these enzymes are used to interconvert purines. So it’s a catabolic pathway mixed with interconversion pathway. We can convert AMP to IMP with deaminase. We discussed that deaminases hydrolyze the side chain amino group off as ammonia. This is an example of a nucleotidase to take AMP to adenosine. This enzyme is possibly important in muscle. It has high activity in muscle.

Adenosine deaminase>extremely important reaction. He said to put an x or star by the reaction*****(adenosine to inosine) Once we have inosine it can go to hypyoxanthine utilizing phosphorylase which uses phosphate and produces ribose-1-phosphate and now we have hypoxanthine.

GMP can be converted to Guanine using nucleotidase (purine nucleoside phosphorylase, guanine deaminated xanthine) so we can call fall down to the oxidation state of xanthine. It’s not what we excrete. When we get rid of purines we get rid of them at the next oxidation level above xanthine.

Guanine and xanthine are the same oxidation level. Go up one more oxidation level and hypoxanthine to xanthine using xanthine oxidase, and we need another reaction also catalyzed by xanthine oxidase to convert xanthine to uric acid. Xanthine goes to uric acid, which is the final product that we excrete.

***XANTHINE OXIDASE>STAR IT. MANY PEOPLE ARE TREATED WITH XANTHINE OXIDASE INHIBITORS.

This is the catabolism of purines.

There’s another pathway called the salvage pathway for purines, but in this case I’ll show hypoxanthine and guanine and there’s one for adenine too, but it requires phosphoriboso-pyrophosphate when our body has expended lots of energy making these rings, so perhaps I don’t want to throw them away and I let them be oxidized to uric acid, I want to salvage them, so you take the free base and react with PRPP to form the monophosphate and in the case of xanthine it’s IMP and with guanine it’s GMP. These are the salvage enzymes.

What type of patient will you see that has an overproduction of purines?

None with have an underproduction of purines because it’s incompatible with life. Several types of patients will have overproduction. Gout! It’s making a dramatic comeback because it’s one of the correlates to the Syndrome X or the metabolic syndrome Extrome X which has high b.p. and high lipid in blood, obesity, etc. It’s called the metabolic syndrome, syndrome X or extrome. Gout is associated with this also.

What type of uric acid levels do you have to have in serum to have gout? Gout, we don’t know why, most of it is indopathic, so we don’t know why it occurs but it’s treatable. If you have less than 6 mg/100 mL of serum uric acid concentration you’re protected from gout. Above 9 or 8-9, most people (he mumbled) and men are very susceptible.

Uric acid in gout is supersaturated, so it begins to precipitate. The serum can’t take that level of uric acid. It varies from patient to patient. If you look at the normal distribution of serum uric acid levels, in normal people vs. “gouty” people there’s some ovelap. Why do these people have gout and others are normal? We don’t know. Once serum-uric acid level is about 8, an individual will typically have a good chance of having gout.

(My comment: Untreated gout looks very sad an painful). He showed pictures of the gout individual. Uric acid precipitates in the joint space. It then elicits an immune response. It allows immunological response to destroy joint space and even bone is eroded.

The therapy for gout.

You’ll all see patients who have had treatment with a xanthine oxidase inhibitor called allopurinol. It really looks like hypoxanthine with N’s in different places. This is oxidized to a small extent by xanthine oxidase. It’s not only a competitive inhibitor but also a competitive substrate. This is what your patients will use it to treat gout.

Graph shows the normal range for serum uric acid. Once you treat patients with allopurinol, most people fall within the normal range of serum uric acid. It’s a very treatable syndrome. Gout is making a strong comeback.

Lesch-Nyhan (shows picture of the boy with lips eaten away and the destroyed finger)

Lesch-Nyhan syndrome is not treatable. It’s fluoride overproduction of purines and uric acid. Purines have to go somewhere. If you make a lot, they have to be metabolized to uric acid. They can’t just be secreted that way. A bizarre hallmark is the self mutilation of the body. The lips have eaten away and the finger has been destroyed. It’s an aggressive kind of mutilation so that you don’t want to get near one of these people because they could take a bite out of you. It is one of the most bizarre metabolic syndromes or inborn errors of metabolism and is typically asked on boards, and we’ll see what the enzyme defect is. It teaches us about regulation of purine nucleotide biosynthesis which we talked about.

The clinical course involves almost all mutilating themselves, and almost all are aggressive. They all have high levels of uric acid. They usually die of kidney disease because the uric acid deposits in the kidneys. They also havegrowth and mental retardation. He showed the normal amount of uric acid index for creatinine, and they’re way above normal. They excrete major amounts of uric acid in urine.

What’s the problem with Lesch-Nyhan(LN) patient?

They have a defect in the hypoxanthine guanine PRPP transferase. This is the enzyme that takes hypoxanthine and guanine and then react with PRPP to form IMP and GMP.

Hyp + PRPP  IMP

Guan + PRPP  GMP But in these individuals, the reaction doesn’t go.

You can actually look at the residual amount of enzyme (it’s a log scale) or activity in LN patient, and it’s usually below 1%. He proceeds to talk about the graph (I would have included the title if I could have figured out the title-sorry). Others are below 1% or some don’t have spasticity or growth retardation. Others have high levels of uric acid. Most gout is idiopathic. You don’t know what causes it. In a small number of patients you do have a known enzyme block. It shows that the enzyme and some of the mutations will produce an enzyme with no activity and others with some activity so that it’s not one mutation of the enzyme. These people are a collection of mutations of the enzymes… it’s not just one single one.

So, what does it tell us about regulation of purine biosynthesis?

We have aminotransferase(first committed step in purine biosynthesis denovo) and we have GMP,GDP,GMP,IMP… that all feed back to inhibit this enzyme. So, if we can make these compounds plus the di- and triphosphates of them so we go on to make GDP and GTP, what are these products going to do to the first step of purine biosynthesis? You have glutamine, ATP and PRPP  NH2-R-P (that represents a chemical equation)… these are going to (they’re at the end of pathway) feed back to inhibit the pathway. Once you get to NH2, the pathway is a little unregulated and goes on to form more purine. LN patient can’t salvage (HYP) to make (GMP) to inhibit that reaction with glutamine. So their pathway runs wide open (which is bad) and if they can’t do this, if they can’t salvage these, (Hyp, Gua) they have to go to uric acid. For LN patients, two things are wrong: without salvage enzyme there’s no feedback inhibition of the first step of the purine biosynthesis pathway so it’s wide open. Secondly, if you can’t salvage Hyp and guanine, they only have one other place to go> they’ll be catabolized to uric acid. It’s a vicious cycle, making more uric acid and letting purine nucleotide biosynthesis pathway make more uric acid> It feeds on itself! These patients tell us something about the regulation.

As long as you have the salvage enzyme operating, they can feedback to inhibit the aminotransferase, preventing the formation of the phosphoribosylamine or the ribose phosphate amine and thus slowing down the pathway.

You’ll see cancer patients treated with methyltrexate, but you’ll see a whole slew of patient who have autoimmune disease. You won’t be tested on this. Low dose methyltrexate for autoimmune disease has taken off since 1990’s.

What is autoimmune disease?

It’s a huge collection of diseases when the immune system attacks itself. So, what can our inhibitor dihydrofolate reductase do? How can it help someone with autoimmune disease? This also illustrates something about purine nucleotide biosynthesis that’ll be asked on boards also.

The bubble boy: This little boy in Texas lives in this sterile environment. Everything in and out is autoclaved. The kid has no immune system. This is several years ago, and they let this kid stay alive so he could be researched. He has no immune system. None. I mean, zero immune system. When he was eleven he was let out and died. This individual has no activity of adenosine to inosine (enzyme: adenosine deaminase<he has no activity whatsoever). So, there’s no possibility that he could metabolize adenosine to inosine. So adenosine builds up in blood and urine. Adenosine is toxic to the immune system. Why is too long of a story. High levels of adenosine shut down the immune system.

Adenosine Deaminase:

3. Deficiency of this enzyme produces severe combined

immunodeficiency disease (SCID)

4. Accounts for 50% of all SCID

5. Plasma and urinary adenosine levels increase in the

deficiency disease

How are they toxic to the immune system?

Here the IL-2 receptor is upregulated during the immune response. This is mouse blood T cells. They stimulate mouse T cells to mount an immunological response. It’s an invitro in test tube experiment. So they put in something to make it mount an immune system T response. It shows the percent of cells positive for the IL receptor. After eight hours you have about 80% of cells positive for that receptor. This is a normal response for mouse T cells.

IL-2 is called an autocrine. The IL-2 is produced by the cell, expressed extracellularly, bound to a receptor and bound to a receptor on the cell membrane. It stimulates itself to proliferation and an immune response. An autocrine is expressed outside of the cell to be bound by a receptor on a cell membrane. It stimulates itself or acts upon itself.

What does adenosine do? As the adenosine level goes up, we can look at the fraction positive for the IL-2 for a few hours. As the adenosine concentration goes up from 1 to 50 micromolar, the number of cells for the positive receptor goes down to almost zero. If you don’t have the receptors you can’t do this. If we have adenosine in plasma, these receptors aren’t formed. Somehow the cell knows not to. It’s toxic to the cell and the cell doesn’t synthesize those IL-2 receptors.

What does this have to do with autoimmune disease?

Let’s look at one enzyme in purine nucleoside biosynthesis that we want to focus on besides the first enzyme. Remember AICAR, which was towards the end of purine nucleotide biosynthesis, and this AICAR needed a one C fragment from 10-formyl-THF and we would synthesize then the final product de novo, which is IMP. If we treat patients with methyltrexate, we know that somehow it blocks AICAR from going to IMP (blocks the enzyme). Therefore, AICAR builds up. From the riboside, once it’s dephoshphorylated, we get AICA-ribose. AICA-ribose inhibits adenosine deaminase. So, if we treat someone with methyltrexate we block that step in purine nucleotide biosynthesis then AICA-ribose increases and blocks adenosine deaminase. Blocking adenosine deaminase is bad for immune system. People with autoimmune disease want something less than a really good immune system. This therapy down regulates to allow their autoimmune system to be controlled. We did research looking at the amount of this particular metabolite in urine before and during methyltrexate therapy. This is in patients with serosis. Most of the patients had an increase in the AICA substance in the urine after dosage with methyltrexate. This person had a gigantic increase (others had great increases). We knew this would happen, but we didn’t know that along with the increase in AICA excretion there was increase in adenosine excretion also. The ones that didn’t have an increase in adenosine excretion didn’t do well with methyltrexate. The drug wasn’t helping them much.

This person had the enormous increase in adenosine excretion and thus had the best overall result with this therapy. We can come up with a mechanism for the treatment of the autoimmune disease with low dose methyltrexate, and it’s really low dose methyltrexate. Distinguish it from high dose methyltrexate, which is used in cancer patients (low dose: MTX 10-20 mg per week for people with autoimmunediseasebut for cancer patients undergoing chemotherapy it would be much greater.) So, we did see both an increase in AICA and adenosine in urine indicating that they were building up in the body, indicating that if this builds up then there’s down regulation of the immune system.