Causes and mechanisms of methaemoglobinaemia

This chapter is probably at least somewhat related to Section F8(vii) from the 2017 CICM Primary Syllabus, which expects the exam candidates to be able to "describe physiology and consequences of abnormal haemoglobin". The principles underlying the detection of methaemoglobin are discussed elsewhere, as are the properties of methaemoglobin. This chapter focuses on the reasons as to why one might have ended up with oxidized haem iron, and how to get out of such a situation.

As with all such "why is there too much of substance x" questions, the answer is usually either excessive synthesis, or ineffective clearance. Both mechanisms need to be explored.

In the interest of rapidly offering a brief summary to those intolerant of tangential gibberish, the following table lists the major culprits:

Causes of Methaemoglobinaemia

Physiological normality Normal rate of autooxidation: 0.5-3% of total Hb per day Direct Oxidants of Haemoglobin Nitrites Sodium or potassium nitrite Amyl nitrite Nitrates GTN Nitroprusside Antimalarial quinones Pentaquine Primaquine (maybe) Chloroquine (big maybe) Methylene blue

Congenital metabolic defects Cytochome b5 deficiency Cytochrome b5 reductase 3 deficiency Haemoglobin M disease Indirect Oxidants of Haemoglobin Aromatic hydrocarbons Aniline Naphthalene Nitrobenzene Sulfonamides Trimethoprim / sulfomethoxazole (Bactrim) Dapsone Random antibotics Nitrofurantoin Local anaesthetics Benzocaine Prilocaine

Constant physiological generation of methaemoglobin

Oxygen itself is (unsurprisingly) an oxidising agent, and at normal atmospheric concentrations of oxygen there is a routine daily rate of haemoglobin-methaemoglobin conversion, which is roughly 0.5-3% of the total haemoglobin pool per day.

Oxygen, when it is normally carried by haemoglobin, oxidises the iron to its ferric (Fe³⁺) state, forming a ferric-superoxide anion complex (Fe³⁺-O₂^-). When it is being deposited in the tissues, much of the oxygen politely dissociates, leaving the borrowed electron with the iron atom. However, some of the time, a superoxide radical (O₂^-) is deposited instead, taking the electron with it and leaving the iron in its ferric state. In this fashion, there is some ongoing physiological oxidation of iron.

Normal clearance mechanisms

One should make a regular attempt to reduce methaemoglobin, or one will find oneself having an FMetHb of 30% after ten days or so. In essence, any mechanism which regenerates NADPH will give rise to an environment which reduces methaemoglobin.

The key enzyme facilitating this step was at one stage called NADPH methaemoglobin reductase, but it is now known that it is in fact the combined effort of two enzymes, cytochrome b₅ and cytochrome b₅ reductase.

Enhanced spontaneous oxidation of haemoglobin in acidosis

The presence of an acidic environment sponsors the spontaneous generation of methaemoglobin, and inhibits its clearance. This is most noticeable among acidotic neonates, as foetal haemoglobin is for whatever reason more easily oxidised. Additionally, it appears that the clearance of methaemoglobin by reduction back to a ferrous state is inhibited by an acidic pH.

Direct oxidants of haemoglobin

Nitrites

The nitrite ion (NO_2^-) in aqueous solution is a weak Lewis acid, i.e. it acts as an acceptor of electrons.

By accepting electrons (and oxygen) from oxyhaemoglobin, nitrite was thought to enhance the process of autooxidation. The reaction has previously been represented in the following manner:

2O₂Fe^{2 +} + 3NO₂^- + 2H⁺ → 2Fe³⁺ + 3NO_3^- + H₂O

That would have been very easy, but subsequently it has emerged that the mechanism is more intricate. The reaction actually appears to be autocatalytic; the presence of methaemoglobin seems to act as a catalyst for the oxidation of oxyhaemoglobin. This has been confirmed experimentally, by tracking the rate of reaction between oxyhaemoglobin and nitrite with spectrophotometry. The graph below is an adaptation of the unreferenced Figure 1 from the 2005 article by Kim-Shapiro et al.; the oxyhaemoglobin was being incubated with 600 micromoles of nitrite at 37°C.

A model which explains this autocatalytic behaviour can be described by the following fusillade of equations, based on the work of Doyle (1982). The incorporation of hydrogen peroxide is called for because it was found that catalase and superoxide dismutase (peroxide-scavenging antioxidant enzymes) interfere with the autocatalytic reaction, straightening the humped concentration-time curve presented above.

Additional support for this hypothesis comes from the experimental evidence that adding hydrogen peroxide to the solution accelerates the reaction by a greater degree than would normally be expected (as H₂O₂ is known to oxidise methaemoglobin on its own).

Unfortunately, there is little experimental evidence to support the last two reactions. In fact there may be little actual haemoglobin-nitrite interaction, and the whole process may be driven by a methemoglobin-catalyzed peroxidase oxidation of nitrite, with the product of oxidation then converting oxyhaemoglobin directly to the methaemoglobin-peroxidase complex, which then goes on to oxidise more nitrite. Or, it may be something completely different. A possible model is proposed in a recent Nature article from 2013. Rather than reproduce their complex diagrams here and try to explain them, one might instead seek shelter under references, specifically detailed references regarding the role of nitrite in nitric oxide biology (nitric oxide, NO, being one of the intermediate species produced in the reaction of NO₂^- and deoxyhaemoglobin). Even having said that, my references themselves run and hide under other references. "Its complicated!" confesses a typical haemoglobin researcher in Kim-Shapiro's textbook chapter from 2005. The chapter's authors, after several pages exploring the history of nitrate-haemoglobin research, lament: "The major conclusion of the above discussion is that despite the fact that the reaction between oxyHb and nitrite has been known for over 100 years, its mechanism still remains elusive".

So.

Nitrites cause methaemoglobinaemia, because smnurh.

A whole host of nitrates are available, which will dissociate into (R) and (NO_2^-) in solution.

For instance:

• Sodium nitrite (meat preservative)
• Potassium nitrite (meat preservative)
• Ammonium nitrite ( a fertiliser- humans are exposed by drinking contaminated farm well water)
• Amyl nitrite and alkyl nitrites in general ( GTN-like vasodilators with a thousand uses)

Of these, sodium and potassium nitrites are probably the most ubiquitous (being present in cured meats, such as salami and bacon). Fortunately, with food safety guidelines, at a concentration of about 2.5-6 ppm, one would have to ingest a truly absurd quanity of bacon in order to be affected- apparently a normal adult lethal dose of sodium nitrite is about 2g . Having said that, idiotic accidents will continue to happen. The BMJ presents a case study of two previously well boys who suffered methaemoglobinaemia after their father fed them home-made sausage marinaded in an unexpectedly concentrated sodium nitrite. In spite of the fact that the children’s father "had been concerned about its quality, as the meat turned green rather than the usual red colour" the sausages were eaten anyway.

Nitrates

The nitrite ion (NO_3^-), the conjugate base of nitric acid (HNO₃) is mentioned here because it is reduced into nitrite by gut bacteria, and then absorbed. In this fashion, one might find oneself unexpectedly awash with an unsolicited excess of nitrite anions. Fortunately, these substances are rarely found within arm's reach of the modern-day man. The 1951 monograph by Bodansky mentions such ancient relics as ammonium nitrate (an old-school diuretic, introduced in 1926, and now more frequently seen in the hands of farmers and terrorists who tend not to ingest it) and bismuth subnitrate (given orally in vast quantities as a radioopaque contrast medium). For those possessed by a tearful nostalgia for these molecules, I can recommend a 2008 review article by Butler and Feelisch from Circulation.

More modern examples are available. Glyceryl trinitrate (GTN) is a common-as-mud vasodilator workhorse of the world's coronary care units; enthusiastic overuse of it can potentially cause methaemoglobinaemia in predisposed individuals . In one case report, a poor 58-year-old NSTEMI patient was infused with 30 μg/kg of it for six days, which was not a super-huge dose (locally we would usually tolerate a maximum of about 200 μg/kg) Unfortunately, the man had some sort of congenital NADPH-MetHb reductase deficiency, and the physicians of the Medical Centre Hospital of Vermond were alerted to this fact when during his CABG the anaesthetist measured a FMetHb of 9.6%. Similarly, nitroprusside use can cause methaemoglobinaemia, which is good of it (to generate its own antidote). In general, in routine practice it seems that it is rare to end up in methaemoglobin-related trouble from the non-ridiculous use of vasodilator nitrates.

One should probably mention nitrofurantoin here. It is not directly oxidative, but it does happen to be photolabile; the photodegradation products include nitrate, which then goes on to generate nitrite, and the rest is predictable.

Chlorates

Again, this is likely a historical footnote from the Annals of Old-School Toxicology. For instance, Bodansky mentions potassium chlorate mainly because of its (in them days) ubiquitous use as the major active ingredient of "mouth washes, gargles and dentrifices". One has little to go on in terms of contemporary experience. The reasons for its fall into disuse may include the fact that it has a tendency to spontaneously ignite or explode, even when mixed with materials which one would not normally describe as combustible. Similarly, sodium chlorate (a nonselective herbicide) is toxicologically apocryphal; its danger to humans these days seems to be restricted to the Mythbusters team.

Quinones

Antimalarial quinones are certainly known to produce methaemoglobinaemia, but mainly in people with enzyme defects. On their own, and in metabolically normal people, drugs like chloroquine and primaquine are probably not going to be responsible for a clinically significant methaemoglobinaemia.

More recent (1994) insight reveals that older agents (pentaquine and menadione) caused marked methemoglobin production in vitro, whereas more modern agents (atovaquone, daphnetin, and menoctone) did not. Some blame falls on the metabolism of otherwise benign substances into highly oxidative nightmares like benzoquinone and napthoquinone.

Methylene Blue as an oxidising agent

The methylene blue dye has the amazing capacity to generate methaemoglobin in the normal organism, and to reduce methaemoglobin in an organism which suffers from its excess. This bizarre property deserves to be explored in greater detail. In fact, methylene blue deserves an entire chapter dedicated to its weird properties, not the least of which are its cardiovascular effects. In the interest of curtailing the already rampant tangentiality of this chapter, I will try to focus only on those properties which are relevant to methaemoglobinaemia.

Specifically, methylene blue seems to act as an oxidising agent in the absence of satisfactory intracellular glucose, and in the presence of vastly ridiculous amounts of methylene blue. Exactly how much is ridiculous, depends on the author. Some say that for haem oxidation to occur doses in excess of 4mg/kg are required. Goldfranks' Manual of Toxicologic Emergencies (2007 edition) recommends to keep the dose under 7mg/kg. A BMJ case report doubles the maximal dose again, to 15mg/kg.

In any case, it seems in the presence of a normal amount of glucose, the normal dose (eg. 1-2mg/kg) will reduce methaemoglobin. To compare, a typical dose of methylene blue which is required to manage severe vasoplegia is also about 1-2mg/kg.

Indirect oxidants of methaemoglobin

Aromatic hydrocarbons: aryl amino and nitrogen compounds

These horribly toxic compounds have a role to play in toxicology, as causes of occupational methaemoglobinaemia. There is a vast range of them, and it does nobody any good to list several pages of chemicals. Among them, the most common culprits are aniline (a solvent) and naphthalene (a moth repellent still occasionally included in mothballs). These families of molecules are better known as carcinogens, and much has been written of the ill effects of long-term exposure to them; only occasionally do they arouse the interest of the haemoglobin toxicologist, and then under extremely weird circumstances (eg. when they are used as a part of the soap used in pediatric enema solutions).

Bodansky, to whose opus I constantly refer, makes mention of about twenty of these molecules, and offers references to researchers who have used them to induce methaemoglobinaemia in cats. One can estimate the cat bodycount from the number of references (at least ten papers are listed). However, no mechanistic explanation of the indirect oxidative effect is mentioned. For the majority of these compounds, that remains the case. It is known, however, that the majority of them do not directly oxidise haemoglobin - instead, hepatic metabolism convertes the inert precursor into something dangerous (eg. aniline into phenylhydroxylamine). A 1999 article offers the suggestion that these metabolites then go on to produce superoxide and peroxide, which in turn do all the oxidative damage.

Sulfonamides

As these drugs are no longer in routine use, this digression again appears to be of purely historical interest. These days, only Bactrim (trimethoprim/sulfamethoxazole) is likely to be of interest, and with its routine use the risk of methaemoglobinaemia is low. That said, case reports are available.

Into this category, I shall place Dapsone, even though it is a sulfone. Dapsone itself is not especially active as a haemoglobin oxidant, but its enterohepatic metabolism produces two long-lived hydroxylamine metabolites, which each have a potent oxidant effect. Thus, dapsone has a dose-dependent effect on the production of methaemoglobin, which usually does not get out of control. For proper methaemoglobinaemia, this case report of a pregnant woman who took a dapsone overdose is probably more representative.

Local anaesthetics

Benzocaine is known to cause methaemoglobinaemia. In one case report, 20% bezocaine throat spray was used to topicalise the the airway, with subsequent cyanosis and some considerable anaesthetic anxiety. Prilocaine is also listed in the usual alphabetised list of drugs which cause methaemoglobinaemia. Like with most of these pharmacological causes, the true mechanism is a matter of educated guesswork.

Congenital metabolic defects as causes of methaemoglobinaemia

Clearance mechanisms for methaemoglobin may be failing in people with a deficiency of the responsible enzyme. There are several enzyme systems which - if broken- might result in methaemoglobinaemia, among other things. For instance, congenital NADH methemoglobin reductase deficiency has given rise to a population of congenitally cyanotic Appalachian hillbillies, who now have a blog. In general, there are several possible disorders which may give rise to a congenitally raised methaemoglobin level (and thus to compensatory polycythaemia). UpToDate does this topic some justice, and I direct their paying customers there for a detailed overview. Destitute freegans must make do with informative case reports and review articles.

Thus, here is a brief and uninformative list:

• Cytochome b5 deficiency
• Cytochrome b5 reductase 3 deficiency (it comes as Type I or Type II)
• Haemoglobin M disease

Agents for the reversal of methaemoglobinaemia, and their mechanisms

Methylene blue as a reducing agent

As already discussed, among the normal methods of disposal for the daily burden of methaemoglobin is reduction though the metabolism of glucose(17%), of which about 5% happens by the action of NADPH-dependent methaemoglobin reductase. The presence of methylene blue can increase the clearance of methaemoglobin by this latter pathway 40-fold.

Observe: by cycling through its two states (methylene blue and leucomethyene blue) this molecule burns through glucose to reduce Fe³⁺ to Fe²⁺ by donating electrons to the ferric iron. Those electrons are in turn borrowed from the metabolism of glucose, via NADPH. Cytochrome b₅ and cytochrome b₅ reductase catalyse this process; it just so happens that these enzymes have an affinity for blue dyes, such as methylene blue, Nile blue and divicine.

Obviously, in the absence of G6PD doing all the heavy lifting with the glucose, none of this will work, and haemolysis will ensue. In such cases, Vitamin C is the answer.

Ascorbic acid as an alternative reducing agent

It appears that ascorbate is a poor second to methylene blue in terms of reaction rate. In dogs, for a dose of around 200mg/kg the same decay of methaemoglobin (FMetHb from 60% to 30%) takes up to 5-10 times as long to accomplish (100-200) minutes versus 20-40 minutes with methylene blue.

The effect of ascorbate appears to rely on the activity of glutathione. However, no sensible-looking mechanism is available anywhere for this reaction. Otherwise detailed reviews skim over this issue, offering only the tidbit that glutathione and ascorbic acid are minor players in the pathway of direct endogenous reduction of methaemoglobin. Sodium ascorbate continues to be co-administered with methylene blue in these patients.