Eliminating Serum Amyloid A as a potential Biomarker in Long Covid
...following a murky, unclear path through to some clarity.
Last week in the piece on biomarkers for Long Covid, we saw that Serum Amyloid A (SAA ) was elevated in the small study of Long Covid patients, making it a potential candidate for a biomarker. Here was the data:
SAA Reference Range: 0-10 mg/L
Mean SAA Concentrations in the study
Long Covid Patients: 6.9 mg/L
Healthy Control Group: 5.3 mg/L
The mean SAA value in the Long Covid patients was higher than the healthy control group by a statistically significant amount (p<0.05).
We also saw that SAA is strongly linked with inflammation. Here’s the information we reviewed last week:
SAA is a protein produced during the acute phase of inflammation. SAA localizes to the site of inflammation and recruits immune cells. The activation profile of SAA is similar to CRP (C-Reactive Protein) and SAA levels increase quickly in the acute phase of inflammation.
This week I started looking more closely at SAA, armed with nothing but questions about its general role in inflammation and its specific role in Long Covid.
Here are some of the questions that immediately popped into my mind:
What is SAA as a protein? Does it have enzymatic function?
How is SAA activated as part of the inflammation response?
How do SAA levels in the blood increase as part of the inflammation response?
What downstream pathways does SAA itself activate, once it’s activated and its levels increase?
What’s known about how SAA is involved in other diseases?
How does SAA interrelate to C-Reactive Protein (CRP)?
What can all of this tell us about the inflammation that’s occurring in Long Covid?
For the rest of this piece, I’m going to work on answering these questions with information from the scientific literature. The information in the answers comes from the literature publications in the References below.
This piece is by design and necessity going to be about exploring SAA, as opposed to trying to tell a complete story. My hope is that by the end, we will have enough information to ask the next set of questions about inflammation in Long Covid to take a step farther down the path of assembling the complete picture we desire.
So let the the Q&A begin:
What is SAA as a protein? Does it have enzymatic function?
SAA is a small protein consisting of 104 amino acids. It’s known as an “apolipoprotein”, which is a protein that will bind to lipids (aka fats) to form lipoprotein complexes. In this case, the SAA apolipoprotein binds to high density lipoprotein (HDL, as in HDL cholesterol) in the blood plasma.
SAA is purely a structural protein and it does not have any enzymatic activity. It does, however, have function as a signaling molecule, binding to receptors and other proteins to transmit biological signals and activate downstream pathways in the inflammation response.
There are four isoforms of SAA (SAA1-4) that are produced by four separate genes (SAA1-4). The two isoforms that are relevant to inflammation are SAA1 and SAA2. These two proteins differ by only 7 amino acids and are generally treated as interchangeable as part of the inflammation response. For the rest of this piece, I will refer to SAA1 & SAA2 together as “SAA”.
SAAs are made predominantly in the liver, but can also be synthesized by cells at sites of inflammation, including in the walls of arteries, lung tissue, brain tissue, muscle, cartilage, adipocytes (fat) cells, skin, and intranasal tissue.
How is SAA activated as part of the inflammation response?
SAA is what’s called an “acute phase protein” that is induced as part of the “acute phase response” to environmental insults including infection, trauma, stress, or tumor growth. The acute phase response is a highly orchestrated and evolutionarily conserved defense mechanism in vertebrates.
The initiation of the inflammatory acute phase response is predominantly driven by the endogenous cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α.
These cytokines then induce the expression of acute phase proteins, including SAA1, SAA2, and C-reactive Protein (CRP, more on that later).
Once expression is induced, SAA is synthesized in the liver and then released into the bloodstream.
How do SAA levels in the blood increase as part of the inflammation response?
Once synthesized in the liver, SAA is released into the bloodstream, so that it can localize to sites of injury and inflammation.
Activation of the acute phase response by an environmental insult causes a 100- to 1000-fold increase in SAA concentration, with maximal blood plasma concentrations reaching as high as 1 mg/mL (= 1000 mg/L). The overall serum concentration of SAA in healthy individuals without inflammation is ~1-2 µg/mL (= ~1-2 mg/L).
SAA concentration data can vary widely from study to study. Studies have been carried out to determine the effects on SAA plasma concentration in many disease states by comparing healthy patients to those with the particular disease.
If we compare these literature reference numbers for SAA concentrations at baseline (~1-2 mg/L) vs. during the acute phase response (as high as 1000 mg/L) to the numbers from the Long Covid patients in the Biomarkers study, we see that the Long Covid patients (mean [SAA] = 6.9 mg/L) and the health control group (mean [SAA] = 5.3 mg/L) have mean SAA concentrations that are the same order of magnitude.
From this, I conclude that:
The Long Covid patients in that study are NOT experiencing inflammation as a result of the acute phase response.
Due to this fact, SAA is NOT a viable biomarker for Long Covid.
There is some data to show that SAA might be a good biomarker for acute Covid infection. But I just don’t think it’s going to work for Long Covid, because the inflammation in Long Covid seems to be chronic and not from acute phase response.
Despite the fact that SAA, its minimal increase in concentration in Long Covid, and it’s mechanistic pathways look like a bust as a potential mechanism that’s driving inflammation in Long Covid, I’m going to answer the rest of the questions below for completeness and in case the information is of interest to some.
What downstream pathways does SAA itself activate, once it’s synthesized and its levels increase?
Going through the scientific literature, there are a number of proteins and receptors that are activated by SAA.
Here I want to focus on two that are important for inflammation: the formyl peptide receptor 2 (FPR2, a G-protein coupled receptor) and Toll-Like Receptors 2 and 4 (TLR2 and TLR4).
Activation of FPR2 and TLR2/4 on monocytes (a type of white blood cell) at the site of an injury or infection helps to recruit other cell types (a process known as chemotaxis) to that site to repair the injury as well as activate the immune system to fight infection.
Activation of TRL2/4 receptors also releases additional IL-1β, IL-6, and TNF-α cytokines that propagate the inflammation response and act as a positive feedback loop.
And activation of TRL2/4 receptors also induces the CXCL8 and CCL3 chemokines.
CXCL8, also known as IL-8, is a signaling molecule that binds to the CXCR1/2 receptors and is a key mediator of inflammation that recruits neutrophils to the site of inflammation.
CCL3 is a signaling molecule that binds to the CCR1/4/5 receptors and is involved in the acute inflammatory response by recruitment and activation of leukocytes (white blood cells).
Here’s a figure showing activation of SAA and subsequent downstream pathways:
Identifying these types of pathways that could potentially be functioning aberrantly in Long Covid as part of the inflammation response was what I’d been trying to do for a few weeks.
But now based on the fact that SAA concentrations are not sufficiently high in Long Covid patients for the acute phase inflammation response to be in effect (see above), it’s time to start looking at other mechanisms which do not involve the acute phase response.
What’s known about how SAA is involved in other diseases?
SAA has been proposed to be a key signaling molecule in multiple disease states including rheumatoid arthritis, obesity, type-II diabetes, atherosclerosis, pulmonary diseases (including COPD and asthma), and cancer.
High levels of SAA proteins are found in the joints of rheumatoid arthritis patients, in blood vessels with atherosclerotic lesions, and in many types of solid tumors.
Published studies have associated tissue‐derived SAA with the progression of rheumatoid arthritis, atherosclerosis, and tumor metastasis.
How does SAA interrelate to C-Reactive Protein (CRP)?
C-reactive protein (CRP) is also an acute phase protein that is generated during acute phase inflammation reaction. Both SAA and CRP concentrations increase rapidly following an environmental insult like trauma or infection. And they also share some intracellular signaling pathways. Both are induced by IL-6 and both are predominantly synthesized in the liver.
While the concentrations of these two proteins mainly move in parallel during inflammation in various disease states, there are cases in inflammatory rheumatoid arthritis where SAA concentration is elevated but CRP concentration is not.
At the same time, increased CRP concentrations with normal SAA concentration “are extremely rarely observed.”
What can all of this tell us about the inflammation that’s occurring in Long Covid?
Based on the fact that, in the study with Long Covid patients, the mean SAA concentration for that group was roughly the same as the mean SAA concentration in the healthy control group (6.9 vs. 5.3 mg/L), it seems safe to conclude that the acute phase inflammation response is NOT active in Long Covid patients. If it were, the mean SAA concentration in the Long Covid patients would need to be many-fold higher (2-fold, 10-fold, even 100-fold higher or more) than that of the healthy control group.
So if the acute inflammation pathways are not active in Long Covid, we will have to start examining the chronic inflammation pathways.
That will be the next set of questions I ask and start answering. So stay tuned!
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Reference
Structure and Expression of Different Serum Amyloid A (SAA) Variants and their Concentration-Dependent Functions During Host Insults. Current Med. Chem. 2016, 23(17), 1725–1755. https://doi.org/10.1189/jlb.3VMR0315-080R.
Serum Amyloid A in Inflammatory Rheumatic Diseases: A Compendious Review of a Renowned Biomarker. Frontiers in Immunology. 2020, 11, 631299. https://doi.org/10.3389/fimmu.2020.631299.
Emerging functions of serum amyloid A in inflammation. J. Leukoc. Biol. 2015, 98(6), 923–929. http://dx.doi.org/10.2174/0929867323666160418114600.