Ever since nitric oxide (NO) was discovered to relax mammalian blood vessel walls, the list of physiological reactions and organisms that this diminutive molecule is found in has grown and grown. Inspired by the discovery that, in mammals, nitrite acts as a NO donor and that deoxygenated haemoglobin(deoxyHb) catalyses this reaction, Frank Jensen from the University of Southern Denmark took the opportunity to look at the role of nitrite in zebrafish, showing for the first time that NO is formed from nitrite in a lower vertebrate (p. 3387).
Inside the red blood cell, deoxyHb reacts with nitrite to form NO and metHb, a form of haemoglobin that can't carry oxygen. The NO inside the cell can then itself tightly bind to more deoxyHb, forming nitrosylhaemoglobin(HbNO). This form also can't carry oxygen, and is also quite stable, so can be used as a biological marker to measure NO levels inside the fish.
Jensen exposed fish to 3 different nitrite levels in their water –none, 0.6 mmol l–1 or 2 mmol l–1 –which they took up across their gills. He then measured the levels of the different haemoglobins (deoxyHb, oxygenatedHb, HbNO and metHb) in their blood at 0, 1 and 2 days after exposure, and also after 5 days in fish exposed to 0.6 mmol l–1 nitrite. Using a spectrophotometer, Jensen measured the amount of light absorbed by each diluted blood sample. Because each of the four haemoglobin types absorb different wavelengths of light, he was able to use this information in a mathematical model that calculated how much each haemoglobin contributed to producing the recorded absorbance spectra. From this he could then calculate the proportion of each of the haemoglobins in the fishes' blood.
Nitrite exposure clearly led to the production of high levels of NO from nitrite in zebrafish, indicated by a major increase in HbNO production. Jensen also notes that partially deoxygenated haemoglobin would have played a role in this process, because haemoglobin generally only becomes partially desaturated in the blood carried in the veins.
Looking more closely at the effect of the different nitrite concentrations,Jensen found that 2 days of 2 mmol l–1 nitrite exposure reduced the total concentration of haemoglobin in the blood samples,suggesting a decline in the number of circulating red blood cells. Of the haemoglobin present, non-oxygen carrying metHb accounted for 59% of the total haemoglobin, and HbNO levels reached 12% of total, reducing the oxygen carrying capacity in these fish to around 30% normal levels. Only some fish in the 0.6 mmol l–1 nitrite exposed group had higher metHb and HbNO levels, with HbNO levels stabilising at around 4%. This shows that upon exposure to nitrite, there can be a big decrease in oxygen-carrying haemoglobin.
Because the oxygen carrying capacity is much lower in nitrite exposed fish,Jensen predicted that this would lead to a reduction in their oxygen consumption. But when he measured the oxygen consumption of fish in a respirometer he found that there was no change. He suspects that if the fish are in a relatively resting state, then nitrite levels don't pose too much of a problem, however this could change if the fish are stressed, for example if they need to escape from predators. Then, higher nitrite levels could mean the difference between life or death.