Fish and Human Studies

June 29th, 2021

This site has been password protected since its creation date of May 18th 2020 when the Covid-19 pandemic was still in the early stages.  Around half of the US population is fully vaccinated.  Even Fox News is passing on a World Health Organization recommendation that fully vaccinated people continue to wear masks and social distance. These ad nauseum news bits are hitting news media like a hurricane The reason for the same old same old is that not everyone is vaccinated.   A vaccinated person may have some plasma cells programmed to produced Covid-19 spike protein antibodies hiding away somewhere in the bone marrow.  A few airway epithelial cells may still be hijacked by this delta variant of Covid-19 before the plasma cells start making antibody again. Masks and social distancing are for everyone!

One does not have to look very far to see that WHO recommendations are being ignored in much of the US. Vaccinated or not, we still need to do our parts to make sure our immune systems are ready. Those who are throwing caution to the wind have even more reason to discuss with their physicians if their diet contains enough copper, niacin, and other minerals and vitamins to maintain proper immune function.

Back in May of 2020

Mitosynergy has a lot of data showing a decrease in neutrophils and an increase in lymphocytes in fish and humans consuming  cuprous nicotinate, Cu(I)NA2,  as a dietary ingredient.  Fish and human studies showed a decrease in neutrophils and an increase in lymphocytes.   Perhaps Cu(I)NA2 could have value as a medical food for patients with one variety of sepsis or another.  Indeed Cu(I)NA2 was originally tested to protect farm fish from bacterial infections.  Thoughts on copper and the prognostic value of the neutrophil to lymphocyte ratio (copper and NLR) have been posted in another blog.

Can Cu(I)NA2 assist the immune system when COVID-19 challenges?

2021 disclosure: Over the past year the neutrophil to lymphocyte continues to be regarded as a good predictor of Covid-19 outcome. We still do not know if a lack of balance causes severe Covid-19 or is merely a symptom Data from the original report are presented as is the role of copper in immune function. Dietary supplements may still be a relevant conversation to have with a physician until the day we each heard immunity .

Chuan Qin (MD PhD) and others published demographic data collected from patients experiencing severe and non-severe symptoms of COVID-19. These data represent 452 confirmed cases of COVID-19 from Tongji Hospital, January 10 to February 12, 2020. Of these, 286 cases were coded as severe, the remainder, non-severe. Blood samples were collected and analyzed. The data included peripheral lymphocyte subsets. Neutrophils were found to be increased in severe COVID-19 infections. Lymphocytes were found to be decreased in severe COVID-19 cases when compared to non-severe cases (Qin 2020). Obviously, data of these patients before they were infected was not available. These data do suggest that the innate immune system might be overshadowing the antibody producing adaptive immune system. This publication has prompted Mitosynergy to go back to old clinical trial data from an otherwise healthy population experiencing chronic pain. Routine blood cell workups were conducted to show lack of toxicity. Cu(I)NA2 trial suggested a significant decrease in neutrophils as well as a significant increase in lymphocytes. Qin and coworkers (2020) suggest that the innate immune symptom might be out of control in severe cases of COVID-19. Data are presented that Cu(I)NA2 might enhance the adaptive immune system that involves antibody production.

Cu(I)NA2MedFood4One thing that is striking about these data is that even the lowest value for neutrophil percentage of total leukocytes in severe cases (69%) is almost more than the highest value in the normal range (75%). The lowest % lymphocytes per leukocytes in normal individuals is 20%. The highest % lymphocytes seen in severe cases of COVID-19 was 21% in the Qin (2020) study.

neutrophil to lymphocyte infection with COVID-19
The neutrophil to lymphocyte ratio found in COVID-19 patients suggested mild stress

The neutrophil to lymphocyte ratio (NLR) is a general bio-marker of inflammation. Qin and coworkers explored this index as a means of differentiating between severe and non-severe cases of COVID-19. We will go through each row and relate the comparison of severe and non-severe cases of COVID-19 to the blood work that was part of a Cu(I)NA2 clinical trial and some fish studies at doses of 30 and 60 mg Cu(I)NA2 per kg of feed.  The skeptic might regard the NLR as so much biomarker propaganda just because it seems to be gaining traction as a diagnosis for every malady known. EMCrit,.org reviews literature regarding the NLR. Qin (2020) did not report the normal range because we don’t know it. The murky gray region might be a normal range in response to an infection.

Leukocytes

Leukocytes is another name for white blood cells comprised of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Qin and coworkers found a significant increase in all leukocytes in severe compared to non-severe cases of COVID-19. All counts were within normal ranges. These authors did not comment further on this particular statistic. The clinical trial of Cu(I)NA2 with 48 participants in the middle of a Canadian winter failed show any difference in the leukocyte count in response to the copper or the elapsed time in the study. A dose dependent increase in the total leukocyte count was seen in mullet (Abo-Ghanema 2007) and sea bass (Saad 2003)

Neutrophils

Neutrophils are a type of first responder phagocytic cell. In bacterial infections neutrophils migrate towards the site of inflammation. Neutrophils typically range from – 2.0–7.0×10 9/L or 40–80% of all leukocytes. Neutrophils also generate reactive oxygen species super oxide, hydrogen peroxide, and hypochlorous acid, better known as bleach (Winterbourn 2016).

Qin and coworkers reported a significant ( p<0.001) increase in the neutrophil count in severe compared to non-severe cases of COVID-19. These authors devoted much of their discussion to the neutrophil to lymphocyte ratio. Note that some in severe COVID-19 group had neutrophil counts that exceeded the normal range.

A dose dependent decrease in neutrophils was seen in mullet (Abo-Ghanema 2007) and sea bass (Saad 2003) In the Mitosynergy human study Cu(I)NA2 decreased neutrophils.

Neutrophils in viral infections and bacterial infections release reactive oxygen species into the phagosome. Mitosynergy Cu(I) decreases neutrophil counts in non-infected subjects
Left  *Between group comparisons were made using two sample independent Student t-test. δWithin group comparisons were made using the Wilcoxon Signed-Ranks test. Probability values ≤0.05 are statistically significant.  Right Neutrophil phagocytosis and oxidative burst to kill bacteria

Is there evidence in the literature that copper affects neutrophils? A previous study (Cintia 2016) demonstrated no change in phagocytic activity of neutrophils from copper deficient versus supplemented cattle in response to a bacterium that causes eye infections. This study was restricted to neutrophils in bovine eye fluids. Differences in superoxide dismutase activity were observed bovines made copper deficient versus the ones that were supplemented. Superoxide could be helpful in scavenging superoxide that escapes from the neutrophil phagosome.

COVID-19_SOD1
Cu/Zn superoxide dismutase detoxifies the reactive oxygen species superoxide.
Lymphocytes

Lymphocytes consist of T cells, B cells and natural killer (NK) cells. T and B cells are part of the acquired immune system that produces antibodies. Natural killer cells are part of the innate immune system. Qin and coworkers subdivided different types of lymphocytes coming to the conclusion that COVID-19 seems to attack T cells in particular. For simplicity, only significant (p<0.05) data are shown from Qin (2020) Table 3.

QinTable3

This is only an abbreviated version of Table 3 showing only significant differences between non-severe and severe cases. Qin and coworkers discussed the importance of T cell subsets in dampening the innate, pro-inflammatory, immune system. Other interesting references were cited regarding helper T cell function.  Note the CD8+ sub population of T cells.   Tarasenko (2017) created a mouse model that lacked the cox10 subunit of cytochrome C oxidase.  Mitochondrial dysfuntion resulted in T cell distributin abnormalities in these mice, particularly in response to a vaccine (Tarasenko (2017).   T cell sub populations was measured with flow cytometry.  Mitotracker Green was used to measure mitochondrial membrane potential.    These assays might be a bit expensive in a clinical trail.

Cytochrome C oxidase activity  in T cell pellets  was measured by a  colorimetric change in reduced cytochrome C.  (Tarasenko 2017).

COVID-19_AdaptiveImmune

Nothing to the best of our knowledge has addressed the possible mechanism of Cu(I)NA2 in antibody production and pagocytic activity.  Does Cu(I)NA2  act on CD8+ versus CD4+ or influence IL-2 or IL-4 signalling?  We know from two fish studies (Saad and  Abo-Ghanema) that there is an increase in both antibody production and phagocytic activity.

A mechanism by which Cu(I) can push the neturophil to lymphocyte ratio in favor of lymphocytes
*Between group comparisons were made using two sample independent Student t-test. δWithin group comparisons were made using the Wilcoxon Signed-Ranks test. Probability values ≤0.05 are statistically significant.

A mechanism?

We simply do not have any solid explanation as to why Cu(I)NA2 would increase the lymphocyte numbers. Supplying the copper to the leukocyte vascular adhesion protein /amine oxidase C3 (VAP-1/AOC3) is a curiosity worth examining.  More down upstream Cu(I)NA2 affects  have been examined in another blog.

Dunkel and coworkers (2014) investigated the role of VAP-1 in recruitment of eosinophils and CD4+ T cells in allergen stimulated mouse airways. The authors compared normal mice and mice that lacked the AOC3 gene, and hence the VAP-1 protein. They found that after tracheal exposure of the allergen, a recruitment of antigen‐specific CD4+ T cells to draining bronchial lymph nodes was reduced by 89% on day 3 in VAP-1 deficient mice. This difference was not observed on day 6. VAP-1 is a member of the semi-carbazide sensitive amine oxidases that utilize copper in their reactive centers. We presently do not know how enzymatic activity might affect CD4+ T cell function. We further do not know if VAP-1 has a tendency to become Cu deficient.

The Fish studies

2021 disclosure: We have come to find out that the US FDA does not recognize fish studies. Some customers may appreciate use of non mammalian models. It is best to discuss this matter with your physician.

These are data from two studies testing the hypothesis that Cu(I)NA2  in the feed protects farm fish from the aquatic pathogen Yersinia ruckeri. The authors performed some basic blood work as well as measuring phagocytic activity and antibody production. Fish were immunized with inactivated Yersinia ruckeri. These data demonstrate the ability of Cu(I)NA2  to boost immune function.

Capital letter: Means within the same row carrying different letter are significantly different at (p<0.05). Cu(I)NA2  increases the antibody titer.
Lowercase letter: indicated that means within the same column carrying different letter are significantly differed at (p<0.05).

Mullet (Abo-Ghanema 2007)

Cu(I)NA2 improves neutrophil to lymphocyte ratio in mullet
Note the increase in lymphocytes and decrease in neutrophils.

Abo-Ghanema (2007) examined the response of the innate immune system by way of phagocytosis of the Yersinia ruckeri bacterium.

COVID-19_mulletPhag
Note the dose dependent increase in phagocytosis of bacteria.

Yersinia ruckeri antibody titers were measured as an index of  Cu(I)NA2 effect on the adaptive immune system.  Fish that had not been immunized had no antibodies against this pathogen.

Cu(I) increases antibody production in mullet
in the feed resulted in a 50% increase in antibody titers against Yersinia ruckeri.

The same group (Saad  2007 )  tested Cu(I)NA2 as a fish feed supplement for farm raised sea bass.  The same modest changes were seen in the leukocyte distribution.

COVID-19seabass
Note the modest but significant increases in lymphocytes and decreases in neutrophils.

Saad (2013) also measured phagocytosis of Yersinia reckeri

Cu(I) improves neutrophil phagocytosis of bacteria in sea bass
The changes in bacteria cleared are significant but modest. There are more bacteria per phagosome in the groups.

as well as antibody titers.

Cu(I) increases antibody production in sea bass
Almost identical small increases were seen in antibody titers.

The results of the second Saad(2013) study were almost identical to the Abo-Ghanema (2007) study.  The second study tested what really matters to fish farmers:  the number of fish that survive Yersinia ruckeri contamination of the water.  This final test put modest, but significant, improvements in numbers into perspective.  Can Cu(I)NA2  improve the ability of immunized sea bass to survive an actual Yersinia ruckeri infection?

Cu(I) is an immune system booster food that helps the sea bass survive actual infections.

All of the fish that had not been vaccinated died.  Twice as many fish in the  60 mg/kg Cu(I)NA2 group survived compared to the vaccinated fish on the control diet.  Cu(I)NA2 improved antibody titer just one weak after vaccination.  These authors did not differentiate lymphocytes into T cells, B cells, natural killer cells, and so on.  As extensive as the Qin (2020) COVID-19 study was, they did not examine antibody producing B cells.  More extensive animal studies of Cu(I)NA2 would allow the examination of  memory B cells that may localize in lymph nodes or Peyer’s Patches.  Plasma cells are the antibody producing B cells that tend to reside in the bone marrow.

2021 parting words…

We think  Cu(I)NA2 has a lot of potential as a supplement to fight infections. Hopefully this post has given you a lot of information to share with your physician as to why copper is important to our immune systems.    

References

Abo-Ghanema I, Saad TT , El-Bahrc SM, Fayza AIE(2007) Some Studies on the Effect of Copper (I) Nicotinate Complex on the Immune Response and Some Biochemical Parameters of Mugil cephalus Fish Vaccinated With Yersinia Ruckeri Bacterin J Arabian Aquaculture Soc. 2(2)105-130

Cintia PG, Leonardo M, Israel OR, Andrea S, Beatriz VL, Elena DM.(2016) Superoxide Dismutase Activity, Hydrogen Peroxide Steady-State Concentration, and Bactericidal and Phagocytic Activities Against Moraxella bovis, in Neutrophils Isolated from Copper-Deficient Bovines. Biol Trace Elem Res. 171(1):94-100.

Dunkel J, Aguilar-Pimentel JA, Ollert M, Fuchs H, Gailus-Durner V, de Angelis MH, Jalkanen S, Salmi M, Veres TZ. (2014) Endothelial amine oxidase AOC3 transiently contributes to adaptive immune responses in the airways. Eur J Immunol.44(11):3232-9. Link

Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y, Xie C, Ma K, Shang K, Wang W, Tian DS. (2020) Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin Infect Dis. 2020 Mar 12. pii: ciaa248.

Saad TT, El-Meadawy Z.Kh.Ali MA, Fayza AIE(2013) Some Studies on the Effect of Copper (I) Nicotinate Complex on the Immune Response and Some Biochemical Parameters of Sea Bass Fish Vaccinated With Yersinia Ruckeri Bacterin World’s Vet. J. 3(4): 74-81

Tarasenko TN, Pacheco SE, Koenig MK, Gomez-Rodriguez J, Kapnick SM, Diaz F, Zerfas PM, Barca E, Sudderth J, DeBerardinis RJ, Covian R, Balaban RS, DiMauro S, McGuire PJ. (2017) Cytochrome c Oxidase Activity Is a Metabolic Checkpoint that Regulates Cell Fate Decisions During T Cell Activation and Differentiation. Cell Metab. 2017 Jun 6;25(6):1254-12  Link

Winterbourn CC, Kettle AJ, Hampton MB. (2016) Reactive Oxygen Species and Neutrophil Function. Annu Rev Biochem. 2016 Jun 2;85:765-92. Link

Published by BL

I like to write educational websites

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: