Science is full of dogmas. But during the process of discovery, some dogmas face challenges, and the dogmas in nitrogen waste management are no different. As all biology students know, the dogma holds that waste proteins are broken down and excreted from the body in a variety of forms, such as ammonia, urea or uric acid. But each waste product comes with a metabolic cost. Although ammonia is the cheapest nitrogenous waste product, even low levels of ammonia in the environment can be extremely toxic, mimicking potassium ions and poisoning the central nervous system. Only aquatic species can take advantage of the energy savings offered, excreting and diluting their waste ammonia in large volumes of water. But ammonia poisoning is too risky for terrestrial animals; they excrete urea instead, consuming 2.5 ATP molecules for every molecule of nitrogen excreted. Meanwhile, uric acid is a safer, but an even more expensive alternative. So there's the dogma; teleosts excrete ammonia because it's cheap and can be readily diluted for safety, while mammals and other terrestrial creatures tend to produce metabolically costly urea or uric acid, depending on access to water.
And that was how things stood until the late 1980s, when David Randall staged an expedition to Lake Magadi in the Kenyan Rift Valley, with an international team of collaborators. But Lake Magadi is no ordinary lake. At a pH of 9.6-10 Randall knew that the sole species of fish living in the alkaline waters, the tilapia Oreochromis alcalicus grahami, must have overcome the caustic conditions, but how? Suspecting that the fish couldn't excrete ammonia because of the alkaline waters, Randall tested to see if the tilapia might produce urea instead. Sure enough, the fish had activated their genes for the ornithine–urea cycle enzymes, thought to be repressed in all teleost species, and were producing and excreting urea. If tilapia in this remote lake could flout the dogma, could other species contravene held wisdom too?
Since Randall's discovery, several groups have identified teleosts that excrete urea, survive dangerously high levels of ammonia, and some that even seem to thrive on the toxin. With an ever increasing number of challenges to the dogma of nitrogen waste management, it seemed to Rod Wilson from Exeter University, England, that the time was right to bring together a collection of discussions and reports outlining some recent developments in this hotly debated area under the title of `Dogmas and Controversies in the Handling of Nitrogenous Wastes'.
Gulf toadfish pulse urea
Patrick Walsh is fascinated by gulf toadfish and all aspects of their ability to switch from ammonia-based excretion to urea. Toadfish excrete waste urea across the gill, losing up to 80% of their nitrogenous waste during one or two daily pulses lasting up to three hours each. But no one knew why toadfish have opted for this relatively costly alternative when ammonia production is far more economical. Keen to discover why toadfish switch to ureotely, as well as the mechanisms that trigger a bout of urea release, Walsh and his laboratory began investigating these strange fish.
Working with John Barimo, Pat Wright and Shelby Steele, Walsh monitored the enzymes essential for urea synthesis during the fish's early developmental stages and found that even at the earliest stages, eggs and larvae produce ornithine–urea cycle enzymes essential for urea production, with juveniles releasing pulses of urea, just like their elders(p. 2011). And when the team analysed juvenile fish's ammonia tolerance it was more than tenfold lower than that of the adults. Maybe the adults resort to ureotely to protect their young from waste ammonia? Walsh and his colleagues measured the ammonia level in the youngster's burrow homes, and sure enough it was well within the low levels that the youngsters tolerate. But were the fish releasing urea in favour of ammonia to spare their young? Walsh calculated the amount of ammonia that a fish would produce if it switched exclusively to ammoniotely. But even then an adult couldn't produce enough ammonia to poison it's young and justify the cost. Walsh and his team suspect that the fish emit urea pulses for another reason: to prevent predators from tracking their burrows by smell. By releasing urea in pulses while away from home, adult toadfish keep their burrows scent free, and hopefully safe from intruders.
Keen to know how toadfish control this pulsatile urea excretion, Walsh and Danielle McDonald have been following up on the observation that serotonin stimulates urea release from the fish's gills, to try to identify specific serotonin receptors that trigger an event. Exposing fish to receptor agonists and antagonists, McDonald and Walsh found that the serotonin signal was mediated by a class of serotonin receptors known as 5-HT2receptors, filling in another vital link in the complex control system which regulates pulsatile urea excretion in toadfish(p. 2003).
Living with ammonia
Even though ammonia is an extremely unpleasant toxin, some creatures cannot escape it. Trout in captivity experience higher levels than they would roaming free, and air breathing fish tend to accumulate high levels of ammonia while out of their aquatic environment. But both species seem to tolerate the toxin,and trout may even benefit! In an intriguing paper Chris Wood challenges the notion that ammonia's effect is always detrimental(p. 2043).
Based on anecdotal evidence that juvenile trout exposed to low levels of ammonia seemed to grow faster than fish in an ammonia-free environment, Wood set about monitoring the growth rates of young fish exposed to ammonia at sublethal levels. Sure enough, exposure to ammonia concentrations of 70–225 μmol l–1 stimulated the fish's growth over a 10 week period. Wood suspects that the fish may use ammonia from the environment as a nitrogen source to reaminate deaminated amino acids, allowing these fish to recycle waste amino acids that would otherwise be lost and grow more than fish in ammonia-free water on the same diet.
On the other hand, air breathing weatherloaches have adapted to tolerate ammonia in order to survive the threateningly high levels that accumulate in their bodies when their paddy field homes dry out. In a review, T. Tsui and colleagues discuss mechanisms that allow this resourceful fish to tolerate potentially fatal ammonia exposures by reducing ammonia production and converting it into non-toxic compounds(p. 1977). Weatherloaches can also tolerate relatively high ammonia exposures, and lose ammonia by evaporation from the skin. Tsui also describes how potassium channels in the weatherloach nervous system are modified, making them impermeable to ammonia ions and protecting the fish from neurological damage.
Although the majority of fish dispose of nitrogenous waste as ammonia, some juvenile fish are capable of producing urea, and trout are no exception. Pat Wright and her team in Guelph Canada are intrigued by the ornithine–urea cycle in juvenile teleosts, and have recently studied arginase, a key enzyme in the urea synthesis cycle in juvenile trout(p. 2033). Wright and her team identified two isoforms of the protein expressed in many tissues,finding Type I arginase expressed at relatively high levels in the liver. On the basis of their analysis of the trout genes, they suggest that `Type I arginase found in ureotelic vertebrates arose in the common ancestor of amphibia and mammals'.
But even though Wright is making breakthroughs in understanding how young trout excrete both ammonia and urea, Chris Wood knew that there was some controversy over fish using protein as a metabolic fuel source. If they used protein exclusively as their fuel source, the sum of urea and ammonia losses should add up to the total amount of protein consumed and nitrogen lost by the fish. But the sum didn't add up. Were trout losing nitrogen through processes other than metabolism?
Working with Makiko Kajimura, Sara Croke and Chris Glover, Wood quantified other sources of nitrogenous waste, and realised that fish lose up to 21% of their nitrogen either in the form of amino acids from the gills, or by shedding protein in mucus from the body(p. 1993). Which means that measurements of the total amount of nitrogen lost by a fish could significantly over estimate the amount of protein consumed as fuel, whereas selectively measuring the nitrogen loss from ammonia and urea probably underestimates it!
Of course mammals face their own suite of waste disposal problems. Some of the nitrogenous waste products we excrete could become insoluble, causing painful kidney stones. Mitchell Halperin and his colleagues at the University of Toronto, Canada, discuss how mammals maintain sufficiently high volumes of urine production to prevent kidney stone formation, as well as the acid/base regulation problems they face while excreting ammonium ions(p. 1985). The team explain that mammals might face occasions when they fail to excrete enough electrolytes so that their urine levels fall sufficiently for kidney stones to develop.
However, they suggest two ways that mammals could overcome this. They could either increase other osmolyte excretion rates to increase their urine output,or under certain circumstances, urea could itself play an active osmotic role to increase urine flow rate, and prevent the formation of kidney stones. Moving onto the problem of acid/base balance, Halperin points out that for ammonium ion excretion, urine should maintain a low pH, but at the risk of allowing insoluble uric acid to precipitate and block the terminal nephron. However, he explains that this conundrum could be solved by NH4+/H+ exchangers in the medullar collecting ducts that could maintain the urine's pH while excreting ammonium, and`minimize the risk of uric acid stone formation'.
Urea counter balance
Of course, some creatures produce urea for reasons other than waste management. Elasmobranchs synthesise high levels of urea to prevent dehydration in their ocean environment. However, while urea production in mature elasmobranches is well understood, less is known about osmoconformation management during the earlier stages of life. Pat Wright, Paul Yancey and Shelby Steele wondered whether little skate embryos produce key components of the urea synthetic cycle and regulate urea levels in response to varying seawater concentrations (p. 2021). Sure enough, even at 4 months the developing embryos produce significant levels of four key urea synthetic enzymes, and regulate urea levels by releasing urea when salt concentrations fall. So, even before they hatch, skate embryos are well prepared for the osmoregulatory challenges they will face in life beyond the egg.
Although these challenged dogmas and controversies will probably see further additions and alterations in the coming years, for many species the dogma of waste nitrogen excretion still holds true. And the creatures that don't conform? Well they'll keep integrative physiologists occupied for several generations, teasing apart the details of the exceptions that prove the rule.