Vitamin D, COVID-19 and Me: Stress Proteins in COVID-19: Heme Oxygenase

Stress Proteins in COVID-19: Is Carbon Monoxide formed by Heme Oxygenase important in drug therapy?

Food for thought for doctors and nurse practitioners who treat COVID-19.

Kanji Nakatsu, Professor Emeritus, Pharmacology, Queen’s University

I spent many years working on heme oxygenase (HO), specifically drugs that inhibited it and some that may have activated it. Examination of COVID-19 (C19) research findings for over a year, revealed many indications that HO might play a role in the disease and that this enzyme could be exploited for treatment.

There are two functional Heme Oxygenases: HO-1 is inducible and is widely distributed whereas HO-2 is constitutive and found mostly in nervous tissue and the testes (1).

While learning about C19 and its treatments, one couldn’t help but be impressed with the large number of different drugs with widely-varying chemical structures that can be used to treat this disease. Even after consideration of the various stages of C19 in which drugs were being used, I was still impressed with the range of drugs that MDs around the world were using successfully. Moreover, the structures of these drugs did not share a common scaffold, which is often the case for medications in the same drug “family.”

Eventually it dawned on me that several drugs used to treat C19 are also inducers of HO-1: they cause an increase in the amount of this enzyme. The following are a small sample of C19 drugs chosen to illustrate the diversity of structures; one feature common to them is the ability to effect an increase in the amount of HO-1 protein.

Background: What is HO?

HO is an enzyme that degrades heme arising from hemoglobin and other hemo-proteins as shown in the figure below.

The co-enzyme of HO is cytochrome P450 reductase. The substrate heme is pro-oxidant while the products are generally antioxidant (2). Interestingly, this reaction results in the formation of carbon monoxide (CO), which is one of three so-called gasotransmitters. The other two are nitric oxide (NO) and hydrogen sulphide. At high concentrations the toxicity of CO is well-known but at low concentrations it is a signaling molecule with a plethora of actions in animals and humans. It regulates the function of a variety of tissues including immune, respiratory, reproductive, gastrointestinal, kidney, and liver- see review (3).

CO anti-inflammatory activity. The pattern of C19 disease has been documented by numerous international groups and is generally recognized to start with a viral multiplication phase followed several days later by an immune reaction phase as illustrated in the 2021 Sep 26 review by Marik et al. (4) below. By the time patients report to a clinic, the viral replication phase is often approaching its end and an immune response, with possible immune dysregulation and exaggerated inflammation, commences. Interested readers should access the original article for full details. Therein, they state: “It is likely that the balance between viral inoculum size, rate of viral replication, the host production of interferons, and pro-inflammatory mediators that determines the outcome of infection with SARS-CoV-2.”

By the time a doctor or nurse sees the patients, usually little can be done to address viral replication and treatment addresses the immune response to the virus and its debris. Clearly, anti-inflammatory steroids have a role, and their judicious use has been remarkably effective in both inpatient and outpatient settings. Practitioners the world over who are accustomed to out-of-the box thinking have identified other treatments; the effectiveness of many of these has been credited to the anti-inflammatory properties of drugs not formally recognized as “anti-inflammatory.” Nevertheless, a common property of these older “anti-inflammatory” drugs is their ability to increase bodily production of CO.

Basic science has revealed that CO has anti-inflammatory properties. For example, Otterbein et al. (5) have described molecular mechanisms by which CO can suppress inflammation. Studies that are more relevant to clinical practice would involve experimental animals in vivo (6). Inflammation of the lungs of pigs was induced by cardio-pulmonary bypass; this resulted in the production of the inflammatory cytokines TNFα and IL-1β. When the animals were administered 250 ppm CO, the inflammatory cytokines were suppressed, and the anti-inflammatory IL-10 was induced (6). This led me to hypothesize that C19 patients who do badly have limited ability to control inflammation due to a compromised ability to synthesize CO. This would fit in with the notion that their ability to deal with stress is impaired relative to patients who are unaffected or minimally affected by C19.

HO-1 is a stress protein- one of a family of stress proteins known as heat shock proteins (HSP). This appellation originated from experiments in which fruit flies were heated to a non-lethal temperature (36°C for 30 minutes); the resulting induced proteins were called heat shock proteins or HSPs. HO-1 is just one of this family of proteins and is also known as HSP32 because of its 32 kDa size. Others in the family are HSP40, 60, 70 and 100 kDa. Subsequently it was found that other stressors also resulted in the induction of HSPs: cold, chemicals, heavy metals, UV light, starvation, hypoxia, infections. This seems to be an ancient response because these stress proteins are found in organisms ranging from bacteria to humans and everything in between.

Only after thinking about a connection between HO-1 and C19 did I search the literature and find papers that were published shortly after the pandemic had been announced (7). Over a year ago, Philip Hooper published an argument for low levels/deficiency of HO-1 being involved with worse outcomes in C19.

“We propose that subjects with metabolic syndrome, old age, and male gender have the greatest morbidity and mortality and have low stress proteins, in particular, low intracellular heme oxygenase (HO-1), making them particularly vulnerable to the disease. Additionally, COVID-19’s heme reduction may contribute to even lower HO-1. Low-grade inflammation associated with these risk factors contributes to triggering a cytokine storm that leads to multi-organ failure and near death. The high mortality of those treated with ventilator assistance may be explained partially by ventilator-induced inflammation. The cytoprotective and anti-inflammatory properties of HO-1 can limit the infection damage. A paradox of COVID-19 hospital admissions data indicates that fewer cigarette-smokers are admitted compared with non-smokers in the general population. This unexpected observation may result from smoke induction of HO-1. Therapies with anti-viral properties that raise HO-1 include certain anesthetics (sevoflurane or isoflurane), hemin, estrogen, statins, curcumin, resveratrol, and melatonin.”

Note that conditions leading to lower levels of HO-1, such as metabolic syndrome, (insulin resistance/diabetes, obesity, hypertension, cardiovascular disease), old age and male sex also lead to worse outcomes when people contract C19. Interestingly, HO-1 is induced by cigarette smoking, resulting in increases HO-1 activity, and increases in blood CO, up to 10% carboxyhemoglobin, mostly due to the CO content of smoke. Hooper also points out that hospitalization of smokers due to COVID-19 was 1/4 to 1/5 that of non-smokers.

Another aspect of HO-1 relevant to C19 is its impact on both innate and adaptive immune responses. Funes et al (8) have described how immunomodulation is affected by HO-1 activity in virtually all immune cells. Additionally, HO-1 induction in mast cells suppresses the degranulation and proinflammatory cytokine production. These authors also provided a list of naturally derived HO-1 inducers: quercetin (flavonoid in fruit/vegetables), curcumin (root of curcuma/turmeric in ginger family), carnosic acid & carnosol (rosemary), resveratrol (wine, cocoa, peanuts), anthocyanins (berries), epigallocatechin gallate (green tea), phlorotannins (brown seaweed), celestrol (traditional Chinese medicine root), caffeic acid phenethyl ester (honey bee hives), capsaicin (chili pepper), garlic-derived organosulfur compounds and isothiocyanates (sulforaphane- cruciferous vegetables like broccoli).

How might HO and CO be exploited in C19?

Doctors and many laypersons are already exploiting various supplements and prescription drugs (off-label) that can increase HO-1 activity in cells. Many dietary supplements (listed above) can act as HO-1 inducers; prescription drugs, such as statins, are also HO-1 inducers. These substances can increase HO activity, and thus CO availability in most cells, but the brain may not benefit because of the distribution of HO-1 in the human body.

As mentioned above, the HO form throughout most of the body is HO-1, except for the brain and testes where the dominant form is HO-2. An important feature of HO-2 is that it is constitutive meaning that its quantity in the brain and testes is not readily increased by various stresses and drugs/nutrient á la HO-1. Nevertheless, my lab showed that it was possible to elevate CO production in brain tissue in test tube experiments by presenting the enzyme with its substrate, heme, in the presence of various drugs including menadione (Vit K3) and supplements, such as riboflavin. Although this work was done using rat brain homogenates, it illustrated the possibility of elevating CO production in the brain by drugs or nutrients (9).

Other considerations.

Substrate supply. A shortcoming that might have to be addressed is the supply of substrate, heme, for both HO-1 and HO-2. One strategy could be the parenteral administration of heme, which is used as hematin for the treatment of certain rare genetic disorders known as porphyrias Another strategy would be to increase the synthesis of heme in situ by administering a heme precursor that enters the heme bio-synthetic pathway after the rate-limiting step. I have seen one report of this by Herman et al. (10) who administered oral 5-aminolevulinic acid to six people; these subjects experienced decreases in blood pressure and increases in heart rate, which is consistent with an increased production of CO with concomitant vasodilation. Various foods (green peppers, bananas, octopus) contain small amounts of 5-aminolevulinic acid, while baker's yeast may contain up to 140 mg/kg.

Turn up the heat. We should question the practice of recommending an anti-pyretic, such as Tylenol/acetaminophen/paracetamol, to patients who test positive for COVID-19. Is an elevated temperature a beneficial response to infections? In many corners, the answer is Yes. My grandmother thought hot baths were good for our health. Look at the "hot" traditions in cultures around the world. In North America, Indigenous Canadians have their sweat lodges; in Europe, the Finns have their saunas and in Asia, the Japanese have their communal hot baths. Experimental animals respond to an elevated body temperature of 40°C for 20 minutes by producing heat shock proteins like HO-1. Should patients take advantage of this innate response to heat stress?

Summary

I wrote this essay because I thought it might facilitate organizing our thoughts of pharmacological/nutraceutical treatment of C19. A role for HO and CO in C19 is far from proven; please feel free to criticize these ideas. When I received my PhD from UBC in 1971, I didn’t think that I would be writing about CO in C19 half a century later.