Introduction: Pulsing LEDs to Activate CCO

This instructable is about how to use sequences of pulses of light in different wavelengths to "resonate" with cytochrome c oxidase in cells to help them generate more ATP with less heat from the LEDs. The light is mainly used for injuries at 4 to 6 J/cm^2 at the cells which means about 100 J/cm^2 need to be applied at the surface of the skin, which can get too hot if a fast treatment is needed (5 minutes). My experience is that pulsing as described here can get the same benefits with really strong and fast pulsing where only 30 J/cm^2 needed to be applied. Constant on devices giving 100 J/cm^2 did not seem to work as well.

There is increasing interest in LED light's ability to penetrate the skull to apply 1 J/cm^2 to the cortex to help neurological conditions, but most serious conditions are not at the cortex level and maybe only 30% of the cortex can be reached with the light due to all its many folds. My method attempts to get a full day of Sun in the effective wavelengths (red and near-infrared, 600 to 900 nm) on a bald head, but in only 15 minutes.

You can use a 555 timer, two resistors, a diode, a capacitor, and an IRF530 mosfet to pulse an LED array down into the microsecond range (which appears to be the optimal range). For example, I have pulsed arrays with cycles of 50 uS on, and 250 uS off. A more complicated circuit will be needed to pulse different wavelength LEDs at different points in the cycle.

The different wavelengths for "healing" are all using the same principle: simply increasing ATP. So there is no theory to support a difference in the quality of the result based on wavelength, only the quantity (830 or 850 nm might be best). However, there seem to be some studies indicating a difference in some qualities like inflammation.

The patentable idea is this: 600 nm to 900 nm LED and LLLT wavelengths can be used to increase beneficial healthful effects with less heating in the skin and circuitry by having pulse-on time of about 50 microseconds and pulse off at least 50 microseconds. I've had great success with 250 microseconds off (17% duty cycle). Ideal pulses might be as low as 5 microseconds "on" to 50 microseconds "off". For even greater effects, different wavelengths can be used together on in a sequence, but still have a primary characteristic of total light on being less than 50% duty cycle.

I can easily see that sequential pulses of 660 nm, 620 nm, and 830 or 850 nm should more efficiently activate CCO than simply constant-on light that I normally promote. ( Not too mention 760 nm which is a hard-to-find wavelength, and when to apply it in the CCO cycle is harder to determine. ) When CCO is pulsed into action with light, then according to the way CCO works as a pump, it will be a while before it is ready for another pulse and therefore more light would be wasted if it is kept on through the whole CCO cycle. Strong light pulses should force most of the CCO's to be in a kind of sync. The duration of the pulses might be most efficient on the order of 1 to 100 of microseconds and the best delays between pulses will be between 20 and 1,000 microseconds. The difficulty is knowing these on and off times, and the sequence of the different wavelengths. Pulses are not an improvement on the way constant-on devices work, except to allow a shorter and deeper treatment with less heat in the skin and electronics. The main problem with LED therapy is that if you apply 180 mW/cm^2 of red and/or near-infrared light, people with white skin get too hot (>105 F, FDA rules) in about 3 minutes which is before the optimal treatment time for beneath-skin injuries (up to 10 minutes). Dark skin will get too hot in 1 minute at 180 mW/cm^2. My personal devices are 180 mW/cm^2 and I move them around or pause a few seconds to prevent too much heat in the skin after 5 minutes. It is a directly proportional correlation between reduced treatment time and the strength of the device. A device with 300 mW/cm^2 applied for 5 minutes appears to have a benefit that is indistinguishable from 30 mW/cm^2 applied for 50 minutes, but heat is a problem if someone where to try to sell such a device, and there is no easy FDA approval process.

Typical LED's that do not need a fan for cooling (and can easily get FDA approval with a letter) emit about 30 mW/cm^2 and seem to have few heat problems even for dark skin (a timer for turning off is needed). So the idea is that since it takes CCO about 10,000 microseconds to complete one pump cycle (averaging 2,500 microseconds per electron, see below), and since it only takes on the order of 10's of microseconds to transfer electrons between the 4 metal atoms, and since it takes only 1 photon per metal atom for activation, then it should be possible to sequence strong pulses and have a long "off" time between pulses, so that the LED circuit and skin do not experience heat, and yet the tissue will get a treatment as if the device were 10 times stronger (assuming a CCO cycle can be sped up from about 10 ms to 1 ms, as my very limited knowledge suugests). In other words, the light pulses can "resonate" with the CCO cycles, speeding them up with less heat. It appears from the CCO pump cycle that the off time(s) can be at least 10 times longer than the "on" times with no decrease in effect from constant-on, so it should be possible to get a 300 mW/cm^2 benefit from a 30 mW/cm^2 device, which means 5 minute treatments for the shoulder and knee compared to 1 hour with a constant-on device that will not reach as deep.

I mention a sequence of different wavelengths, but even 1 wavelength of 620, 670, or 830 nm may be enough (although not ideal), since I know the wavelengths by themselves work, and the CCO complex can't (for the most part) use more than 1 photon of a particular wavelength at a time, and that it is very roughly 50 to 1,000 microseconds before it needs another photon.

My method is to first find out how long human CCO typically takes to complete a cycle. The only number I have is 10,000 microseconds for bovine heart CCO to convert one molecule of O2 (another paper agrees). I begin by dividing by 4 for each of the 4 electrons that need to be transferred from cytochrome c during a cycle as an estimate for how long it is before the next electron is needed. This is really too long because it appears 2 of the electrons are transferred very close together while the rate-limiting step(a) is (are) between the other 2 electrons, but I'll shorten the timing in a later paragraph. I want to give a strong pulse of all three wavelengths so that any spare electrons on each of the 3 metal atoms can be "kicked" to the next step. So the pulses should be about 10,000 / 4 = 2,500 microseconds apart. But I want make the ATP production cycle go faster in the injured cells than what normally occurs in resting healthy cells. I think injured cells and "exercising" cells need to and can produce a lot faster if the nutrients are present and needed, so I'll wildly guess they can efficiently run 10 times faster than normal cells. In other words, instead of 2,500 microseconds between electrons and the needed light pulses, I'm shooting for 250 microseconds. If this is too fast, then some light energy might be wasted, but not nearly as much as constant-on.

Note on the rate-limiting steps: the transfer of an electron from cyt C all the way to the bimetal core can be less than 100 microseconds and yet it takes a 4-electron CCO cycle roughly 10,000 microseconds instead of 400 microseconds. The rate-limiting step(s) could be connected to each other and occuring during the electron transfer between hemeA to the hemeA3/CuB bimetal core, waiting on molecular structural changes in the bimetal core, or waiting on an electron to transfer from cyt C to CuA. See the images below.

I divide my 250 microseconds per electron transfer goal by 10 as the total amount of time I want the light pulse to be on for each "accelerated electron cycle" (25 microsecond pulses, 250 microseconds apart). Short pulses that are only 1/10th as long as the off-stage can be 10 times more powerful in terms of light coming out without burning the LEDs up or heating the skin. The hope is that I can thereby force all the CCOs to be "in sync". I know 30 mW/cm^2 does not cause too much heat in skin or the circuitry, so I should make these pulses 300 mW/cm^2 which is only 30 mW/cm^2 averaged over the cycle. I know from experience 300 mW/cm^2 does not appear to be "wastefully intense", but it should be getting close, so I do not wish to try 20x stonger pulses (and LEDs can't handle it). 50 microsecond pulses half as strong still spaced 250 microseconds apart is also reasonable (shooting for 5x instead of 10x). LEDs are about 25% efficient, so I know my pluses will require 1,200 mW/cm^2 electrical energy.

A 1.5 microsecond pulse every 150 microsecond might be plenty (as they tried in tomato plants at 668 nm for photosynthesis which uses similar biological structures. It seems what I am attempting was tried in this paper in tomatoes and they failed. 10 to 20 microsecond pulses every 100 microseconds may be best because 100 microseconds is about the speed at which 2 of the 4 electrons in a cycle can make it through (the other two are probably much slower, see below). "Kicking" the metal atoms with light creates an electrostatic *pull* on the cyt C, and a *push* on the "bimetal core" where the main enzymatic reaction with oxygen and water is occurring. (( Side Notes: Interestingly, in the plant article above it took 10 photons to convert 1 molecule of CO2 into O2. If every metal atom step of CCO doing the reverse process of converting O2 to CO2 required a photon (850 nm for CuA, 620 nm for heme A, and 660 nm for heme A3 to CuB) then it would take 12 photons (3 wavelengths times 4 electrons). In the plant, 2 millisecond pulses every 200 milliseconds (1,000 times slower) caused photosynthesis to be cut in half. ))

Since 3 LED wavelengths are hard to crowd into 1 unit, I could try 830 nm and 660 nm (or 620 nm). I'll probably try 830 nm by itself since I have the most experience with it being "constant on" and I need to compare. 660 nm instead of 620 because even though it does not have as much absorption, it is uniquely active at the core during the slowest part of the cycle. I'll update this paragraph when I find out how well it works. It takes some time because I have to wait on several good injuries in the family to test it on (I'm writing this June 2012, so maybe I'll know by January 2013).

There are numerous other studies that show a little or no benefit from pulsing, but only a few with < 500 microseconds as required by theory, and none had detailed enough abstracts to see if their duty cycle was correct as predicted by CCO theory. The pulsing I'm describing would hopefully show 5 to 10 times benefit (more cell attachment in test tube) from equal light energy.

Ideally, it might be better to do a *sequence* of < 10 microsecond light pulses of different wavelengths instead of all wavelengths at once. For example, a 660 nm pulse followed by 620 nm, and then 830 nm. This could persuade a reaction at the end stage (in the "bimetal core" with 660 nm, see below), to create an "open slot" (electrostatic pull) for an electron from the "previous" metal atom (heme A) so that the next wavelength can be better utilized to activate (620 nm). The 850 nm pulse would be creating an open slot (electrostatic pull) for the rate-limiting cytochrome c to insert the next electron into CCO. (( BTW, this can cause an electrostatic pull that transmits back up the entire electron transport chain to the other complexes which independently transfer another 4 H+ per CCO cycle. This can prevent electrons from "leaking" and thereby causing free radicals and possibly enable more efficient "fat burning"....if someone exercising at the same time. )) Getting the timing for each pulse and off time is very complicated since there are still too many unknowns in CCO activity. The best off time between the 670 nm and the 620 nm will be difficult to determine, but 620 might be simultaneous with 850. It might turn out to be best to apply all three at the same time because the 2009 article below says the heme a to a3 rate-limiting step may coincide with a cyt c to CuB rate limiting step.

The image above came first because this is complicated. CCO is a set of 13 protiens that accept an electron from cytochrome C and convert O2 to 2H20 while pumping 4 H+ into the intermembrane space for subsequent ATP generation. There are 2 main proteins out of these 13 that are the main "subunits" of action where there are 2 copper and 2 iron atoms that transport the incoming electron, break apart O2 to react into H20 (releasing a great deal of energy), pump the 4 H+, and, very importantly for our purposes, react to incoming light. A copper atom (CuA) accepts electrons from the previous step in the electron transport chain. The previous step in the chain is cytochrom c. The interaction between cytochrome c and CCO is believe by some to be the rate-limiting step, so getting the electron off off CuA into the next step is important to enable cytochrome c to donate the next electron more easily. This atom is responsive to a broad band at 830 nm (i.e., 850 nm works). The transfer to heme A takes about 50 us. This is the speed of transfer, not necessarily including the time the electron is held between transfers. The CCO turnover rate in action is about 10,000 microseconds. I do not know where the main time delay is, but newer paper says O to R and older papers indicated a lot of delay in F to O. Heme A3 shows weaker bands at 610 and 660 nm, where 660 nm was believed to be modulated by the redox states of CuB, and newer paper seem more sure. Note that the chemical absorbances of 600 to 610 seem to need to be about 620 nm "in situ" to get better cell activity. The absorbance of CuB is "invisible" (other than 660 nm) because it is so close to heme A3 (7.5 A). The fact that 660 nm works well indicates that what is absorbed is put to good use, and the fact that it is only active in the O to R time period and considering that time period has a huge delay, it is no surprise. Heme A's absorbsance at 605 nm is more intense than heme A3's 610 and 660 nm absorbances. These electron transfers are reversible which seems to conserve electrons until CCO is ready to pump (which is when enough H+ are in the intermembrane volume, O2 is ready in place at the binuclear center, and assuming cyt c has been supplying the needed electrons). If there are not enough electrons, then more heat is produced in the alternate step (shown in diagram) which takes longer to react (> 600 us verses 150 us according to a 2004 paper). So someones chemistry (fitness) that is good at acquiring the electrons from food energy chemicals can do more work with less heat.

It appears an electron and H+ come into the heme A3 and CuB bimetal core at the same time, roughly being attracted to each other. The pumping action of finally getting "that" H+ to the outer membrane is triggered by another H+ coming into CCO which cancels a negative charge in the bimetal core which releases an electrostatic pull on the "to be pumped" H+ that was being held in place.

When I first get into sunlight, I can immediately see at least a 25% increase in my breathing rate, well before there is any extra warmth. The Sun is providing about 30 mW/cm^2 of the ATP-generating wavelengths, 95% of which is blocked. Maybe only 20% my cells are being exposed (~ 1 cm deep on 1/2 my skin surface). This implies 0.25*20/0.20 = 25 times increase in those sunlight-exposed respiring cells. This increase in breathing stops if I do not engage in exercise and the ATP builds up to block further H+ pumping. This reasoning indicates exposure to these wavelengths while exercising will help increase the ability to exercise. There was a paper showing more fat is burned. Running and lifting weights on the beach is an ideal way to do this, especially if sunscreen has helped prevent the skin from tanning which would block a lot of light. The heat itself should also help the conversions (which is increased by tans). As a 3rd example, I know 10 minutes of strong LED therapy decreases pain for at least 2 hours.

Can I count the number of photons used for ATP generation? At 60 kJ/mole in situ (not 30 kJ/mole) ATP->ADP releases 5E-20 joules per ATP (about 140 moles ATP per day). For a 2,000 kcal diet and 1.75 m^2 body surface area, this turns out to be 5E16 ATP conversions (5E16 electrons) per second per cm^2. Light can reach only about 10% of tissue, so where light can reach there are 5E16 * 0.1 = 5E15 electrons per second per cm^2 needed to produce normal amounts of ATP. 30 mW/cm^2 light has 100E15 photons per second per cm^2, so it has a potential of providing 20 times normal cell ATP production while the light is applied. But about 95% (19 out of 20 photons) is wasted in skin and other absorption. So 30 mW/cm^2 can only bring injured cells up to normal respiration while the light is applied, which I know from experience requires about an hour to see healing effects, and this personal observation is in agreement with the 4 to 6 J/cm^2 research: 30 mW/cm^2 * 0.05 transmittance * 3600 seconds = 5.4 J/cm^2. It is no surprise that bringing injured cells up to normal respiration for an hour would have substantial benefit. But I know from experience on many pains over the past 10 years that 180 mW/cm^2 gets the same benefit in 5 to 10 minutes, indicating that it is possible to speed up injured cell respiration rate by at least a factor of 6.