Greetings, Earthlings! (I’ve got to come up with some better greetings, you know? All of my posts start with, “Hello!”) I hope you’ve been having a smashing week (back to school, if you’re a student with a schedule like my school). I thought I was going to hate it, but it’s actually been pretty good so far, for me!
Anyway, on to the science! Today’s post is going to start us in a different direction than we’ve been going in for most of our discussions on biochemistry. For probably the first time, instead of talking about metabolisms that make energy or store energy, we’re going to talk about metabolisms that get rid of waste products.
Of course, before we can talk properly about how we get rid of toxic wastes, we have to identify them. That’s what today’s post is going to discuss: welcome to Nitrogen, Wild Wolrd of Wonder!
In case you weren’t aware, there’s a lot of freaking protein in our bodies. You might not be feeling it right now (if you’ve just eaten a breakfast of popcorn, like I have), but trust me, you’ve got a lot of protein in you. In fact, 20% of your body mass can be attributed to the stuff.
Now, we’re used to dealing with carbohydrates and lipids, which are all hydrogen, carbon and oxygen. However, amino acids are a whole different ball game: they’ve got lots of other stuff in them that makes them a little more interesting. More specifically, amino acids have amine groups, which contain—shocker!—this lil’ atom called nitrogen.
As you may expect, our bodies pay attention to the amount of nitrogen that goes through them. The biochemistry term for this is that our body cares about its nitrogen balance, which is just the difference in the nitrogen that we take in and the nitrogen that we put out.
Our intake of nitrogen comes from two primary sources. One of them is the source that you’re already thinking of: we get a significant amount of nitrogen from the proteins in our diets, which are broken down into amino acids before they’re absorbed into our blood.
However, there’s another source of nitrogen, as well. That source is a little nastier (in several senses of the word): bacteria in our gut generate ammonia, which is absorbed into our blood, as well.
Now, as you might expect, if you know very much about it, having ammonia in our blood is no bueno. (In fact, in the posts that I’m going to write, you’ll see that it’s not just a Bit Not Good, but, in fact, Very Not Good.) Because of that, our body quickly traps it and gets rid of it. This leads into the four ways that nitrogen is excreted from our bodies: as straight-up ammonia (which is very toxic), as urea, as urate, and as creatinine.
Urea is a much more benign nitrogen-containing molecule than ammonia, and thus, our body sees fit to make it from ammonia in large quantities. All it takes is a little carbon dioxide, some thermodynamic prodding and some enzymatic magic. The others, however, are used in much smaller amounts. Urate comes from purine (adenine and guanine) catabolism. Creatinine is derived from creatine, the energy-carrier of the muscle. If either of the last two are elevated, it’s usually a sign that your kidneys are pulling shenanigans (and by “pulling shenanigans,” I mean “possibly dying”).
In adult humans, we want our intake from food and gut bacteria to be about equivalent to our output in urine. This is called neutral nitrogen balance. However, in children (who are growing—hmm, yes, I just bestowed some knowledge upon you), a positive nitrogen balance is required (wherein more nitrogen is taken in than let out). Negative nitrogen balance is basically always indicative of Bad Things™.
Now, you might ask, what specifically might throw you into such a state of negative nitrogen balance? Well, consider this: our proteins have twenty unique amino acids in them. Of these, our bodies can easily synthesize twelve. That means that, in adults, there are eight amino acids that we must take in, or that are dietary essential: isoleucine, leucine, valine, phenylalanine, tryptophan, threonine, methionine and lysine.
If we’re missing any one of these, our body will start prowling around trying to find them. Don’t you worry, it’ll succeed—it’ll find them in proteins, which it will happily rip apart to get to them. All of those freed amino acids are then broken down and excreted. This ultimately means that you’re putting out more nitrogen than you’re taking in—negative nitrogen balance.
You’ll notice I qualified my above statement with “in adults,” too. That’s because there are two additional amino acids that children must take in from their diet: histidine and arginine. The reason these amino acids are dietary essential only in developing children is exactly what you’d think: children are in positive nitrogen balance, and can’t be bothered to funnel off their nitrogen to make these nitrogen-rich amino acids.
All right, so that’s what happens once we get nitrogen into our body, but how, specifically, do we take it in? Does our stomach lining just swallow proteins whole and shuttle them through our blood? Well, no. That would be kind of creepy.
Instead, when we eat proteins, they denature (lose their shapes) in the acid in our stomach. The bonds between the amino acids slowly hydrolyze in the acid, but our body isn’t content with that. Instead, it sends out pepsin, an enzyme that cleaves the amide bonds of aromatic amino acids, and does it best at pH 2 (stomach pH).
After this stuff reaches the small intestine and is neutralized by bicarbonate, more proteases are released by the pancreas. Elastase cleaves elastin, and trypsin and chymotrypsin cleave amide bonds of aromatic or positively charged amino acids. Carboxypeptidase and aminopeptidase cleave terminal bonds from the carboxyl and amino ends, respectively. Peptidases on the intestinal wall help chew these things up, too. Once they’re broken down sufficiently, they’re absorbed into the blood.
Once they’re in the blood, they’re pulled up by our liver. Our livers selectively metabolize nonessential amino acids (the ones we can make), making them into chemical energy and urea. However, it leaves branched chain amino acids like isoleucine, leucine and valine in the blood—these are a preferred energy store for cells like muscle cells.
All right, now that we know how we get these things into our bodies, let’s talk again about what we do with them once we get them there. (I realize this layout is a little haphazard, but I’m just going off of my notes, so…)
Well, in my previous post on polar lipid synthesis, I showed you that there are a few easy ways that our bodies interconvert different kinds of polar lipids based on what we need. Turns out, we can do that with amino acids too, and we do it using a class of enzymes called aminotransferases.
Aminotransferases, also called transaminases, transfer amine groups from one amino acid to an alpha-keto acid, generating a new amino acid and an alpha-keto acid. Under standard conditions, this conversion is reversible, meaning that the direction of this reaction is really just dependent on what you need. All of these enzymes are dependent on PLP (pyridoxal-L-phosphate), a B6 derivative.
Now, why is this relevant? Well, glutamate is a molecule that is often used as storage for nitrogen. This also means that, when transaminases want to make amino acids, they often use glutamate as a nitrogen source. Here’s a general reaction:
This shows an aminotransferase reaction that uses the enzyme alanine aminotransferase (ALT) to make alanine and alpha-ketoglutarate from glutamate and pyruvate. The enzyme that carries out this reaction is present primarily in the liver, meaning that elevated levels of ALT in the blood are an indicator of liver damage.
A similar transferase reaction occurs between glutamate and oxaloacetate, generating alpha-ketoglutarate and aspartate. The enzyme that does this is called aspartate aminotransferase, and it’s present predominantly in liver, heart, kidney and muscle. Thus, elevated serum AST levels can be a sign of organ damage.
Now, I said that glutamate was a storage molecule for nitrogen, didn’t I? Well, that doesn’t just mean it’s a source of amine groups for amino acids. An enzyme called glutamate dehydrogenase, which is present in most of our cells in both the mitochondrial matrix and cytosol (isoforms), can actually rip the ammonia out of glutamate, making it back into alpha-ketoglutarate. Now, you might be wondering, “Self, why the heck would you want to do that?” Well, if you’re a liver mitochondrion, you find the idea of pulling this off pretty appealing.
Usually, the process of oxidizing glutamate into alpha-ketoglutarate and ammonium isn’t thermodynamically favored (the free energy change is about +20 kJ per mole), but since ammonia is kept so low in our cells, it’s spontaneous under cellular conditions. Thus, using NAD+, we can use this to liberate ammonia for urea synthesis.
(The opposite reaction is technically possible via cytoplasmic glutamate dehydrogenase, in which NADPH is used to assimilate ammonia into alpha-ketoglutarate, but because of the concentration thing, people don’t know if this happens to a great extent.)
Now, once we finally prod our cytosolic glutamate dehydrogenase (liver) into making glutamate, we have another enzyme that goes an extra mile. Glutamine synthetase, an enzyme highly expressed in brain, liver and kidney tissue, uses ATP to combine ammonia and glutamate into glutamine. This is a highly useful process in general, since it produces glutamine, a non-toxic, nitrogen-rich molecule that can be transported safely to other tissues. It’s also useful for the brain specifically, since it regulates both a waste product (ammonia) and a common neurotransmitter (glutamate).
Finally, to complete the whole thing, we’ve got an enzyme called glutaminase. It does exactly what you’d think—it irreversibly hydrolyzes glutamine into glutamate, liberating ammonium in the process. This is useful in the liver, where it starts nitrogen elimination by converting glutamine into glutamate (which will then be converted into alpha-ketoglutarate). However, it’s also useful in the brain, where it’s used to to convert glutamine (used to reload presynaptic ends) to glutamate.
Now, allow me to go on a tangent for just a second. We’ve talked a lot about glutamate and glutamine, but what if we want to convert aspartate into asparagine? Is there an asparagine synthetase that does the same thing as glutamine synthetase? Well, yes and no. There is an asparagine synthetase, but it uses a completely different mechanism to irreversibly convert aspartate into asparagine. Rather than using a single phosphate group from an ATP to convert aspartate to asparagine, it makes an aspartyl-AMP intermediate. What this means, practically, is that it takes twice as much energy to accomplish this conversion.
Asparagine synthetase is active in a lot of tissues besides the liver, unsurprisingly. Also unsurprisingly, an asparaginase enzyme is active in the liver, liberating ammonia from asparagine and making aspartate in the process.
All right, so that’s all of the little pieces. Now it’s time to put it all together. Just humor me for a minute, ’cause I have a feeling that this’ll be useful.
Under anabolic conditions, ammonia is assimilated into nitrogen-carrying molecules that can supply nitrogen for amino acid biosynthesis. Alpha-ketoglutarate is converted into glutamate using glutamate dehydrogenase (NADH) and ammonia. Glutamate can be reacted with an alpha-keto acid to form other amino acids using an aminotransferase (PLP). These amino acids are released into the blood. Glutamate can also be converted into glutamine using glutamine synthetase (ATP) and another ammonia. Glutamine is put into the blood, as well.
Under catabolic conditions, things switch around. Glutamine and amino acids are taken in from the blood. Amino acids convert alpha-ketoglutarate to glutamate using those same aminotransferases (PLP). Glutamine is converted into glutamate using glutaminase (water). Glutamate dehydrogenase then performs its more favorable reaction in the liver, oxidizing glutamate into alpha-ketoglutarate while liberating ammonia.
Here’s where we start to see the big picture: our bodies move nitrogen around rather strategically, sending it where it’s needed when it’s needed without upping blood ammonia levels too much. Now let’s put this in the perspective of actual cellular processes that go on under catabolic conditions.
In muscle tissue, glucose enters muscle cells and is converted into pyruvate using glycolysis, generating energy and NADH. Glutamate is converted into glutamine, trapping ammonia. Alanine is made from pyruvate using ALT. Alanine and glutamine are released from the cell into the blood, where these products are dumped from other kinds of tissues, as well.
Liver cells pick up the alanine and glutamine from our blood. Glutamine is converted first into glutamate using glutaminase, and then into alpha-ketoglutarate using glutamate dehydrogenase. Alanine is converted into pyruvate, ultimately releasing nitrogen while providing pyruvate that can be used in gluconeogenesis. Glucose is released into the blood for respiring cells to consume, and the generated ammonia is funneled into the urea cycle. This is called the Alanine-Glucose cycle, also called the Cahill cycle.
Dang! Who would have thought that nitrogen regulation would be so complicated? It’s further complicated by the fact that there are apparently no good sources of information about this on the internet (Cahill cycle grumble grumble grumble). However, now that we’ve gotten it out of the way, we can look in depth at another metabolism: the biosynthesis of urea.
Questions? Comments? Flames? Links to reliable sources for this stuff? Pls?