cytochrome C oxidase activity deficiency in aging

We found an absolutely beautiful image of cytochrome C oxidase activity in a skeletal muscle section. [1] This site failed to reference the claim that 3% of the muscle fibers in the elderly are deficient in the copper cofactor enzyme cytochrome C oxidase (CCO) enzyme activity. CCO/complex IV terminates the electron transport chain of the mitochondria. Naturally this claim had to be investigated. An internet search was performed for more beautiful images of CCO activity in aged human muscle sections.

• We found an excellent 2011 publication that investigated the increased age related muscle frailty in AIDS patients on long term retroviral treatment. [2] This paper had much to say about the retrovirals accelerating the accumulation of mutations in the mitochondria genome which contains the gene for CCO. A keyword search of the PDF file of this paper for “copper” revealed nothing!
• This year, 2023, an amazing review was published in Experimental Gerentology on muscle aging. [3] “Ultrastructural changes, including loss of type I and type II myofibers and a greater proportion of cytochrome c oxidase deficient” was a major theme, but not one word whatsoever on copper! What? This cannot be!
• Then we found a 2003 paper on the use of copper deficiency to accelerate cardiac muscle aging in a rat model was discovered that explains so much. [4] This research was performed before 2004 when 90% of the human genome had been sequenced. [5] Technology has enabled us to learn so much more, yet we are forgetting the basics. A brief nod may be given to a new 2018 assay to measure CCO activity deficiency in small tissue samples.[6]

Never mind genes, diet induced CCO deficiency and more

The food that these rats was recorded. Then another two groups of ten rats were fed 80% of the rats that were eating as much as they liked. copper-adequate diet (5.0 μg Cu/
g diet) or copper-deficient diet (0.3μCu/g diet) for five weeks after which time their hearts were harvested. Two different different observations were being pursued with this study.

1. Copper deficiency causes mitochondrial oxidative stress by depriving cytochrome C oxidase of adequate copper.
2. Caloric restriction is a good thing.

Over the course of this study the rats grew from about 78 g to about 200 to 270g. The rats eating as much as they wanted of the copper adequate grew significantly more than the other three groups. Food restricted/ copper adequate rats experienced the same growth as the copper deficient rats that were allowed to eat as much as they wanted. Food restricted, copper deficient rats grew significantly less than the other three groups. There was no statistical synergy between copper deficiency and food restriction. The 10% adequate copper in the diet meant about 10% control copper in the liver. Copper deficiency tended to decrease the amount of iron sticking around in the liver.

Multifaceted mitochondrial issues with copper deficiency

Before looking at important “and more” enzymes other than cytochrome C oxidase in complex IV, we need a cartoon.

The Looking for Diagnosis does an excellent and quick overview of how mutations in proteins involved in getting Cu to complex IV can cause production of reactive oxygen species. (ROS). Note the decrease in glutathione peroxidase activity as well as the minochondria’s resident scavenger of superoxide O₂– Manganese superoxide dismuase, also called SOD2. Note the one O₂ picking up one electron e^–. This is a math mistake. The cartoon should have two yellow circle electrons taking O₂ to H₂O. When our bodies make the same mistake we reduce O₂ to the deadly reactive oxygen species super oxide written as O₂ ^●- The “●” is chemistry for a an unpaired electron. Electrons in this life are always looking to pair up.

One of them is blood vessel relaxing nitric oxide (NO) to from reactive nitrogen species peroxy nitrite ONOO^–

Role of superoxide dismutases and glutathione peroxidase

Then we get to our dynamic dual. Both starred superoxide dismutases (SOD) take superoxide back to oxygen and hydrogen peroxide, H₂O₂.

Glutathione peroxidase takes reduced glutathione (GSH) and H₂O₂ and gives us harmless H2O and oxidized GSSG.

Now that we know the dynamic dual, let’s look at data

• where a unit of CCO is the amount that catalyzes the oxidation of μmol ferrocytochrome c / min, For non chemists, they are looking at color changes in ferrocytochrome C per minute.
• a unit of MnSOD is the amount that causes 50% inhibition in the rate of pyrogallol autoxidation, Superoxide likes to react with pyrogallo causing a color change.
• and a unit of GPX is the amount that catalyzes the oxidation 1 nmol NADPH / min.
• The authors did some math so that they could meet the rule requirement to use a statistical technique called the 2-way ANOVA.

enzyme	diet	food restriction	combination of the two
CCO	P=0.0001 99.999% sure	P=0.067 93.3% sure	P=0.0002 99.998% sure
MnSOD	P=0.0006 99.994% sure	P=0.01 99% sure	P=0.01 99% sure
GPx	P=0.0001 99.999% sure	P=0.14 86% sure	P=0.052 94.8% sure
volume of mitochondria	P=0.008 99.2% sure	P=0.002 99.8% sure	P=0.0001 99.99% sure

Even back in 2004 mitochondria swelling was a common symptom of copper deficiency. These are two images of heart cross sections analyzed by electron microscopy. The image on the right was adjusted in a free version of PhotoShop called Gimp2.

What does this mean for humans?

The take home is that a diet deficient in copper for five weeks really decreased cytochrome C oxidase activity as well as activity of anti-oxidant enzymes MnSOD and GPx. The authors didn’t even analyze for Cu/Zn SOD! Johnson and Newman [4] also did not measure copper content in the rats’ hearts. [4] The odd thing is that of the rats with unrestricted food access the final weight was~270 vs ~240 g. How do we know we are getting enough copper? Five years ago an assay was published to detect cytochrome C oxidase activity in muscle (not heart) biopsies of rodents and humans. [6] Dr Leslie Klevay, also of the University of North Dakota, has spent his career studying copper deficiency. Here is a direct quote from a 2022 review’s conclusion. [7]

“One can conclude from numerous medical articles that copper deficiency contributes to, and probably causes, Alzheimer’s disease, ischaemic heart disease, some myelodysplastic syndrome and postmenopausal osteoporosis. These chronic diseases have low organ copper and impaired metabolic pathways dependent on copper. They improve with supplements containing copper. Thus, they exhibit classical characteristics of deficiency.” [7]

The study of Johnson and Newman [4] gives us some onsite on the what might be the cause of multi-organ disease states: loss of cytochrome C oxidase activity and anti-oxidant helpers MnSOD and GPx. [4]

References

1. Frontal Cortex Online Course
2. Payne BA, Wilson IJ, Hateley CA, Horvath R, Santibanez-Koref M, Samuels DC, Price DA, Chinnery PF. Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations. Nat Genet. 2011 Jun 26;43(8):806-10. PMC free article
3. Picca A, Lozanoska-Ochser B, Calvani R, Coelho-Júnior HJ, Leewenburgh C, Marzetti E. Inflammatory, mitochondrial, and senescence-related markers: Underlying biological pathways of muscle aging and new therapeutic targets. Exp Gerontol. 2023 Jul;178:112204. PMC free article
4. Johnson WT, Newman SM Jr. Copper deficiency: A potential model for determining the role of mitochondria in cardiac aging. J Am Aging Assoc. 2003 Jan;26(1-2):19-28. PMC free article
5. National Genome Research Institute
6. Simard ML, Mourier A, Greaves LC, Taylor RW, Stewart JB. A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency. J Pathol. 2018 Jul;245(3):311-323. PMC free article
7. Klevay LM. The contemporaneous epidemic of chronic, copper deficiency. J Nutr Sci. 2022 Oct 11;11:e89. PMC free article