Studies done in trained individuals already make it abundantly clear that very little progress and signs of adaptation occur in response to shifting resistance training-related variables, but regardless, studies do often produce some results that suggest that some degree of muscle size and/or strength gains are indeed possible beyond the first year of training. However, upon closer inspection, some of these results are indeed dependent on the changing variable(s) they are subjected to, and how measures of size & strength are conducted.

The Glycogen Variable

During both aerobic & anaerobic exercise, a muscle’s glycogen reserves dwindle as glycogen oxidation (glycogenolysis) is carried out in endeavors to supply the working tissue with the energy substrates it needs to stay operational, and only after a resupply of carbohydrates in the aftermath of these training sessions do these reserves not only reattain previous stature, but also grow beyond what once was in certain circumstances. But because every gram of glycogen holds at minimum 3g of water, and potentially as much as 17g of water, the more expansive the glycogen reserves of a muscle tissue, the inevitably larger the cross-sectional area of the tissue will be.

Fernández-Elías VE et. al.

Now consider that many studies involve untrained individuals who will obligatorily experience glycogen storage expansion in response to the training they undergo. Consider even the studies that involve trained individuals who are taking on a training protocol that is often very different than what they’re accustomed to, again creating the potential for an obligatory glycogen storage expansion to meet the needs of the body in the face of this new training stimulus. Surely, glycogen metabolism is impacting the results of measurements of muscle size in most Exercise Science studies being pushed out, whether the authors are acknowledging it or not.

References

1. Gentil P, Fisher J, Steele J, Campos MH, Silva MH, Paoli A, Giessing J, Bottaro M. Effects of equal-volume resistance training with different training frequencies in muscle size and strength in trained men. PeerJ. 2018 Jun 22;6:e5020. doi: 10.7717/peerj.5020. PMID: 29942690; PMCID: PMC6016534.
2. Fernández-Elías VE, Ortega JF, Nelson RK, Mora-Rodriguez R. Relationship between muscle water and glycogen recovery after prolonged exercise in the heat in humans. Eur J Appl Physiol. 2015 Sep;115(9):1919-26. doi: 10.1007/s00421-015-3175-z. Epub 2015 Apr 25. PMID: 25911631.

Never RP et. al.

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Variables & Outcomes That Must Be Explored

Increased RT frequency should obligatorily raise glycogen synthesis demands as the muscle tissue seeks increased glycogen reserves to meet the heightened exercise demand. With every gram of glycogen stored, at least 3g of water accumulates², and as such, increased RT frequency should lead to significant water-weight gain by virtue of the expanded glycogen storage. How the body responds to this newfound water load could be critical in determining how bodily testosterone levels are impacted. For instance, in many individuals rife w/ compromised blood vessels, like those w/ varicose veins, varicocele, kidney damage, longCOVID/MECFS, and so on, the body will not allow the water weight to go unchecked, as it poses a threat to the vulnerable & compromised vessels by way of increased blood volume & pressure (water is a major constituent of the blood). If the water-weight & accompanying increased blood volume triggers a resultant compensatory diuresis effect (water efflux) in the body in efforts to protect against the consequences of high blood volume & pressure, total blood volume will decline. As total blood volume declines, hematocrit, a measure of the percentage of hemoglobin in the blood, will automatically be rendered elevated, as the denominator in the calculation (total blood volume) has declined. To compensate for these now elevated hematocrit levels, the body should theoretically limit testosterone availability, be it by increased SHBG & other binding agents, be it by increasing testosterone metabolism, or be it by decreasing testosterone production. Why? Because as I’ve exhaustively detailed everywhere across this site, testosterone is intimately connected to hemoglobin & hematocrit measures;

Since testosterone is critical to muscle hypertrophy, this presents a scenario wherein increased training frequency would limit testosterone bioavailability, thereby limiting the muscle’s hypertrophic potential despite theoretically increased muscle protein synthesis as a consequence of increased bouts of resistance training.

References

1. Neves RP, Vechin FC, Teixeira EL, da Silva DD, Ugrinowitsch C, Roschel H, Aihara AY, Tricoli V. Effect of different training frequencies on maximal strength performance and muscle hypertrophy in trained individuals-a within-subject design. PLoS One. 2022 Oct 13;17(10):e0276154. doi: 10.1371/journal.pone.0276154. PMID: 36228016; PMCID: PMC9560172.
2. Fernández-Elías VE, Ortega JF, Nelson RK, Mora-Rodriguez R. Relationship between muscle water and glycogen recovery after prolonged exercise in the heat in humans. Eur J Appl Physiol. 2015 Sep;115(9):1919-26. doi: 10.1007/s00421-015-3175-z. Epub 2015 Apr 25. PMID: 25911631.
3. Yarrow JF, McCoy SC, Borst SE. Tissue selectivity and potential clinical applications of trenbolone (17beta-hydroxyestra-4,9,11-trien-3-one): A potent anabolic steroid with reduced androgenic and estrogenic activity. Steroids. 2010 Jun;75(6):377-89. doi: 10.1016/j.steroids.2010.01.019. Epub 2010 Feb 4. PMID: 20138077.

A prolonged capacity to increase muscle size & strength in males without sufficient (pubertal) testosterone levels is a myth sold to us by the fitness & supplement industry. It’s a myth that’s been bought into by scores & scores of individuals, and nowhere is this more evident than in IG comments sxn’s, wherein armies of adoring observers rush to defend the natty claims of influencers sporting extraordinary fizeeks, the procurement of which they attribute to years of disciplined training.

The advertised formula for natty success assumes that discipline, married to a significant investment of time, will get you the fizeek you want eventually, and honorably so. You just have to persevere, and over the years, you’ll get your dream fizeek despite abstaining from juice. And while I respect this formula for all that it stands for, this isn’t a Disney production, and not everything plays out this cleanly.

Counts et. al.

Counts et. al. reviewed several studies that measured muscle size at 3 different time-points b4, during, and/or after a resistance training regimen. These studies specifically included presumably post-pubertal individuals, since the inclusion criteria required >17 years of age for all participants. Counts et. al. found that, for one, muscle growth occurred abruptly in the regimen, and for two, muscle growth stalled out relatively soon;

Not only were plateaus evident within 3 months of the commencement of these resistance training protocols, but they developed despite efforts for progressive overload.

Further evidence of the sheer difficulty in ongoing muscle strength & size gains experienced by trained individuals comes in the form of a study carried out by Gentil et. al. in college students (all w/ >/= 1 year of continuous resistance-training experience). The study itself sought to determine the difference that resistance-training frequency would make on both muscle size & strength adaptations when volume is equivalent. Group 1 condensed all their weekly volume into one longer resistance-training session, while Group 2 distributed the workload across two sessions per week. The study found that neither group increased their strength after 10 weeks. Hell, Group 1 even had negative delta values in peak torque (a surrogate for strength).

It’s worth noting here though that Group 1 did experience significantly increased elbow flexor muscle thickness despite the negative delta values for peak torque, but the study authors go on to suggest that the novelty of the training stimulus may have been causative therein;

You see, all study participants had been training each muscle group twice per week for four months prior to the commencement of the study. So while Group 2 simply went on to continue at this frequency, Group 1 instead experienced a novel stimulus that consisted of their weekly volume being crammed into just one longer training session per week. It is tempting to speculate that the increased energy demand associated with accumulating all the volume over one single session would increase the demand for energy storage in the muscle, which would then result in an adaptation wherein glycogen load increased. And since every gram of glycogen associates with at least 3g of water³, this sort of adaptation would indeed trigger a potentially measurable increase in muscle thickness for this particular group, even in the absence of actual myofibril hypertrophy. As such, the results of this study are not suggestive in any way of the strength-capacity-increasing myofibril hypertrophy.

Counts et. al. suggest that muscles are metabolically demanding to maintain, and that’s why plateaus are hit so abruptly after beginning a resistance training regimen;

While this might just be accurate to some degree, I don’t believe this explains it all. I think there’s more to it, that it’s about the evolutionary significance of puberty relative to the rest of an adult’s lifespan. I think humans are wired to become flexible in terms of strength & size during the pubertal, heavy-activity, developmental years, and from post-puberty onwards, with declining testosterone as the cue, the body shifts into strict maintenance mode. No more growth, only maintenance & survival. It’s a theory that insinuates that failure to put size on during puberty means that you’re relegated to a certain ceiling of muscle size & strength forever after. That is, of course, unless you hack your endocrinology with some sauce.

References

1. Counts BR, Buckner SL, Mouser JG, Dankel SJ, Jessee MB, Mattocks KT, Loenneke JP. Muscle growth: To infinity and beyond? Muscle Nerve. 2017 Dec;56(6):1022-1030. doi: 10.1002/mus.25696. Epub 2017 Jun 11. PMID: 28543604.
2. Gentil P, Fisher J, Steele J, Campos MH, Silva MH, Paoli A, Giessing J, Bottaro M. Effects of equal-volume resistance training with different training frequencies in muscle size and strength in trained men. PeerJ. 2018 Jun 22;6:e5020. doi: 10.7717/peerj.5020. PMID: 29942690; PMCID: PMC6016534.
3. Fernández-Elías VE, Ortega JF, Nelson RK, Mora-Rodriguez R. Relationship between muscle water and glycogen recovery after prolonged exercise in the heat in humans. Eur J Appl Physiol. 2015 Sep;115(9):1919-26. doi: 10.1007/s00421-015-3175-z. Epub 2015 Apr 25. PMID: 25911631.

Testosterone has long been known to be increased by exercise;

But the rise itself has consistently been found to be transient in nature, with testosterone levels returning to baseline before a half-hour has even passed in some instances;

It would be too easy to hypothesize that training-induced increases in testosterone were due to the anabolic stimulus that testosterone could provide to the stressed, and soon to be regenerating, muscles as the body strives for adaptation, but in reality, it’s likely much more complicated, and perhaps less glorious, than that. You see, testosterone has a slew of functions in both the male & female body, and one of those functions is to relax blood vessels;

Accordingly, an argument can be made that increased androgenic serum concentrations in response to exercise is directly related to the body’s urgent need to manage the vascular pressure-related stresses that are borne out of these resistance training sessions.

We know that a bout of resistance training will induce significant surges of blood perfusing into the trained muscles. We call this the “pump.” The pressure of this surging blood likely stresses the canals of the vessels which serve as conduits for perfusion, thereby producing a demand for agents that can help manage the pressure. In comes testosterone for one of its probably most inglorious responsibilities: vasorelaxation, which will serve to reduce blood pressure.

References

1. Schwab R, Johnson GO, Housh TJ, Kinder JE, Weir JP. Acute effects of different intensities of weight lifting on serum testosterone. Med Sci Sports Exerc. 1993 Dec;25(12):1381-5. PMID: 8107546.
2. Perusquía M. Androgens and Non-Genomic vascular responses in hypertension. Biochem Pharmacol. 2022 Sep;203:115200. doi: 10.1016/j.bcp.2022.115200. Epub 2022 Aug 1. PMID: 35926652.

Testosterone is intimately involved in red blood cell (RBC) production through the process of erythrocytosis. Testosterone is so influential in the production of RBC’s, that many individuals with elevated testosterone levels, whether natural or not, often present with elevated hemoglobin & hematocrit levels that, at times, breaches the ceiling on standard reference ranges. Indeed, polycythemia, a condition defined by abnormally elevated red blood cells (such that hemoglobin >170g/L, and hematocrit >52%), is considered one of the most common adverse effects of testosterone replacement therapy. A retrospective assessment by Hajjar et. al. found that 24% of subjects receiving 200mg Testosterone Enanthate or Cypionate every 2 weeks (100mg/wk) went on to develop polycythemia. A meta-analysis by Calof et. al., meanwhile, found that men receiving replacement doses of testosterone were 3.67-fold more likely to develop polycythemia.

Hajjar et. al.

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Calof et. al.

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Now consider, we’re referring to individuals who generally have their testosterone dosages prescribed by monitoring physicians in efforts to simply re-establish levels within the normal reference ranges as it relates to their ages. Indeed, the study done by Hajjar e. al. involved only a 35ng/dL difference in mean total testosterone levels between the testosterone-treated & control groups, while Calof et. al.’s meta-analysis involved a more expansive, but still relatively unexceptional, 197ng/dL difference between groups, and yet still, polycythemia was consistently significantly more likely to occur in the TRT groups. Expand our scope, then, to the scores & scores of individuals, both young & old, who utilize androgenic/anabolic steroids (AAS) recreationally to get swole, employing dosages often several fold higher than what would be administered during physician-overseen TRT (for example, 500-1000mg Testosterone Enanthate per week in some advanced AAS users, compared to 50-100mg/wk in many individuals undergoing TRT). It’s easy to speculate that a degree of the disease & suffering, and even loss of life, that we’re witnessing amongst fitness influencers and others who have dabbled in the forbidden juices is a consequence of the androgen-induced polycythemia that, for what it’s worth, no one seems to be paying any mind to.

The Dangers Associated w/ Polycythemia

One prospective study by Braekkan et. al. that examined 26,108 subjects who participated in the Tromso Study of 1994-1995, and amongst who, all first-time life events of venous thromboembolism were documented until 2007, found that men with hematocrit percentages >46% had a 1.5-fold & 2.4-fold increased risk of total & unprovoked venous thromboembolism, respectively, compared to men whose hematocrit were in the lower 40th percentile;

This suggests that even the 54% ceiling that Canadian guidelines use as a trigger for recommended cessation of TRT may be too high.

Venesection (Blood-Letting) as Perhaps the Only Means of Protecting Against Polycythemia

Many physicians & patients interpret the elevated hemoglobin/hematocrit levels in individuals undergoing TRT to suggest that therapeutic phlebotomy (venesection, or blood-letting) should be carried out;

This, of course, would seem a sensible pathway to managing the problem, because a typical blood donation, as an example, involves a donor parting ways with 460-500mL of blood (roughly 10% of their supply), and with that blood goes scores of red blood cells, given that RBC’s make up ~45% of one’s blood supply. However, unfortunately, according to recent findings in males being dosed with replacement levels of testosterone, even routine blood donations aren’t doing enough to abrogate the risks;

Chin-Yee et. al. reviewed the hemoglobin & hematocrit levels of all male blood donors at Canadian Blood Services in Southwestern Ontario who were on TRT, only to find that in 25% of the donated samples, hemoglobin & hematocrit levels were higher than the levels at which Canadian guidelines would recommend a cessation of TRT (hemoglobin >180g/L, hematocrit >54%). Even more concerning was that 44% of repeat donors demonstrated persistently elevated hemoglobin/hematocrit levels above this same ceiling, indicating that, even at presumably modest TRT dosages, the risk of polycythemia remained both persistent & prominent. If blood donations, executed on average every 84 days in this study’s population, weren’t enough to stifle the incidence of polycythemia in TRT patients, then what recourse does someone using/abusing AAS have in managing their RBC counts?

Until this question is answered, we’re bound to continue witnessing more young & old lives alike being lost due to the cardiovascular risks associated with recreational AAS use.

References

1. Braekkan SK, Mathiesen EB, Njølstad I, Wilsgaard T, Hansen JB. Hematocrit and risk of venous thromboembolism in a general population. The Tromso study. Haematologica. 2010 Feb;95(2):270-5. doi: 10.3324/haematol.2009.008417. Epub 2009 Oct 14. PMID: 19833630; PMCID: PMC2817030.
2. Chin-Yee B, Lazo-Langner A, Butler-Foster T, Hsia C, Chin-Yee I. Blood donation and testosterone replacement therapy. Transfusion. 2017 Mar;57(3):578-581. doi: 10.1111/trf.13970. Epub 2017 Feb 1. PMID: 28150363.
3. Hajjar RR, Kaiser FE, Morley JE. Outcomes of long-term testosterone replacement in older hypogonadal males: a retrospective analysis. J Clin Endocrinol Metab. 1997 Nov;82(11):3793-6. doi: 10.1210/jcem.82.11.4387. PMID: 9360543.
4. Calof OM, Singh AB, Lee ML, Kenny AM, Urban RJ, Tenover JL, Bhasin S. Adverse events associated with testosterone replacement in middle-aged and older men: a meta-analysis of randomized, placebo-controlled trials. J Gerontol A Biol Sci Med Sci. 2005 Nov;60(11):1451-7. doi: 10.1093/gerona/60.11.1451. PMID: 16339333.
5. Ohlander SJ, Varghese B, Pastuszak AW. Erythrocytosis Following Testosterone Therapy. Sex Med Rev. 2018 Jan;6(1):77-85. doi: 10.1016/j.sxmr.2017.04.001. Epub 2017 May 16. PMID: 28526632; PMCID: PMC5690890.

This isn’t to claim that you require supraphysiological levels of testosterone, but you likely do require levels similar to that seen during puberty in males if you want to continue making strength & size gains beyond your first 3-12 months of resistance training.

Testosterone Amplifies Satellite Cell & Myonuclei Counts

Muscle hypertrophy/growth depends on new myonuclei entering muscle fibers, and this in turn depends on satellite cells myogenically differentiating (maturing) into myonuclei.

In a vacuum of resistance training, testosterone is still capable of dose-dependently increasing both myonuclei & satellite cell counts, as demonstrated by Indrani et. al.;

So while we’ve all heard the phrase before, it may actually be true; sufficiently elevated androgen levels can facilitate muscle hypertrophy even while the individual is sedentary.

Testosterone Determines How Effective your Caloric Surplus Will Be as it pertains to Muscle Hypertrophy

Influencers will tell you to eat more food, explaining that your caloric intake simply isn’t high enough. But without testosterone-based pressure, the calories consumed in excess of the body’s daily metabolic needs will inevitably face some pressure to become incorporated into fat tissue instead of muscle tissue. Testosterone controls the path that pluripotent stem cells take, and it does this by inhibiting adipogenic differentiation, thereby inhibiting the possibility of said stem cells maturing in ways characteristic of the adipose lineage, while concomitantly triggering myogenic differentiation, such that the stem cells mature along pathways beholden to the myogenic lineage.

Based on this explanation, we understand then that a degree of testosterone’s impact on satellite cell & myonuclei number increases is related to testosterone ensuring that pluripotent satellite cells take the train headed towards new muscle growth.

If you’re capable of comprehending the concepts discussed in the above article on a more detailed basis, help yourself to the “Muscle Hypertrophy” or “Biology/Endocrinology/Physiology” Extensive Notes, which can be found in the Shop. Perhaps you’ll connect dots that I couldn’t.

References

1. Sinha-Hikim I, Roth SM, Lee MI, Bhasin S. Testosterone-induced muscle hypertrophy is associated with an increase in satellite cell number in healthy, young men. Am J Physiol Endocrinol Metab. 2003 Jul;285(1):E197-205. doi: 10.1152/ajpendo.00370.2002. Epub 2003 Apr 1. PMID: 12670837.
2. Yarrow JF, McCoy SC, Borst SE. Tissue selectivity and potential clinical applications of trenbolone (17beta-hydroxyestra-4,9,11-trien-3-one): A potent anabolic steroid with reduced androgenic and estrogenic activity. Steroids. 2010 Jun;75(6):377-89. doi: 10.1016/j.steroids.2010.01.019. Epub 2010 Feb 4. PMID: 20138077.

Often, you read commenters on bodybuilding & biohacking platforms suggesting that simply reconstituting your lyophilized IGF-1 analogs with bacteriostatic water will suffice, and while this may confer peptide stability for some period of time, the extent of that period of time is unbeknownst to any of us due to a total lack of literature on the matter. If a researcher wants a solution that is intended to stabilize the contained peptides beyond just a slew of days, opting for 0.6% acetic acid solution might be more advantageous. This is because the acidity of the solution containing the peptide is critical in determining how stable the peptide will remain. According to Merck, there are at least five potential degradation pathways for peptides, and of these five, two pathways involve base-catalyzed reactions, while another pathway becomes more & more likely as the containing solution grows more basic¹. With that written, it is necessary to note that bacteriostatic water does possess some qualities that do offer some protection to the peptides it will mix with in that respect: it has a pH range of 4.5-7.0, with an average at 5.7, meaning it is slightly acidic (neutral pH is 7, with pH <7 being acidic, and pH >7 being basic). However, a 0.6% AA solution boasts a pH of approximately 2.87;

0.6% Acetic Acid pH Calculation

So comparatively, a 0.6% AA solution is 682-fold more acidic than bacteriostatic water, which could theoretically lead to enhanced stability of the dissolved peptide, granting the researcher more time to consume any given prepared vial. This expanded longevity is demonstrated by an IGF-1 product formulated by Cerilliant that contains a comparable solution acidity (pH = 2.6 in this case, owing to a higher AA concentration of 2%)⁷. Notably, this solution has been demonstrated on analysis to preserve ~97.8% of the native (unmodified) form of IGF-1, and its producers indicate that it is capable of being refrigerated for 8 weeks;

Most researchers will not opt to spend eight weeks dosing out of a reconstituted vial of IGF-1, as most experiments typically last 4 weeks & under. But perhaps confidence can be derived from knowing that a solution only 1.58-fold more acidic than a homemade iteration consisting of 0.6% AA can survive that long in a refrigerator.

Acetic Acid as an Antiseptic Agent

In a experiment carried out by Fraise et. al. involving Gram-positive & Gram-negative bacteria, acetic acid was found to inhibit bacterial growth at concentrations as low as 0.166%, several fold lower than the 0.6% typically utilized to reconstitute lyophilized IGF-1 variants.

Fraise et. al.

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In a study carried out by Trzaska et. al. testing AA’s fungistatic & fungicidal capacities across four species of the Mucorales fungal order (known to induce mucormycosis, which is fatal in >90% of established infections), concentrations of AA as low as 0.3% were found to exhibit potent antifungal effects. The authors were also able to reveal that these antifungal properties are not solely due to the pH of the solution, as other organic & inorganic acids of similar pH did not exhibit as profound an effect.

Trzaska et. al.

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In an in vitro study carried out by Alphin et. al., AA at concentrations of 1 & 3% were found to inactivate influenza on metal surfaces. Separately, in an in vivo study carried out by Pianta et. al., a group of COVID-19-infected individuals treated with 0.34% AA inhalation experienced improvement in symptoms relative to the group that did not receive the inhalation treatment.

Alphin et. al.

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Pianta et. al.

Now, this is not to discredit bacteriostatic water (0.9% benzyl alcohol) as an antiseptic agent in its own right. But every antiseptic agent differs in its effectiveness against the colossal spectrum of pathogens that exist. While a pathogen may exhibit vulnerability to one antiseptic agent, it may, at the same time, demonstrate a degree of resistance to another antiseptic agent. Based on this reality, it may be advantageous to utilize both AA & bacteriostatic water, instead of simply relying on one or the other. Indeed, this is how Increlex, an FDA-approved recombinant IGF-1 peptide, is formulated, albeit utilizing the conjugate base of acetic acid (which should theoretically still display some degree of antiseptic traits, as described by Trzaska et. al.);

Ultimately, reconstitution with 0.6% AA is an exceedingly simple task, even if it adds some steps to the process. This video demonstrates that. Indulge.

Going Above & Beyond; Opting for a Lower-Concentration Reconstitution Solution

While 0.6% AA is about as tried & true as a reconstitution agent can be, there may be room to formulate a lower concentration that could maintain similar antiseptic capacities, while additionally conferring benefits on two fronts: peptide stability and injection pain. We know from references detailed above that 0.3-0.312% AA has been proven to be effective as an antiseptic against a wide range pathogens, including bacteria & fungi. However, the highly resistant MRSA requires a leap up to a minimum concentration of 0.625% for growth inhibition, which is greater than the 0.6% traditionally employed for IGF-1 peptide reconstitution. Based on this detail, any concentration of AA greater than or equal to 0.312% would theoretically inhibit many of the same pathogens that a 0.6% solution otherwise would. Understanding that a lower concentration of AA can maintain antiseptic functionality, we can now explore potential benefits with regards peptide stability & injection pain, and we’ll do this by utilizing a solution of 0.333% AA as an example.

Peptide Stability

Earlier in this article, a lower pH was described as generally being conducive to peptide stability, and this is arguably proven by Cerilliant’s IGF-1 formulation, with a pH of 2.6, that demonstrated 97.8% purity on its Certificate of Analysis, and that allegedly lasts for 8 weeks in a refrigerator. With this written, however, one possible peptide degradation pathway should be acknowledged: deamidation. Deamidation occurs with Asn or Gln amino acid residues, of which, the IGF-1 peptide has 1 & 2 of, respectively. Now, to be clear, deamidation occurs much more frequently in neutral or basic conditions than acidic, as Bhatt et. al. demonstrated in an experiment that compared a range of pH’s & buffer concentrations to determine differences in the rates of deamidation of the singular Asn residue of the adrenocorticotropic hormone (ACTH);

Bhatt et. al.

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Acidic pH is clearly favorable for peptide stability in the face of deamidation pressure when compared to neutral or basic solutions. But what happens to the observed rate of deamidation when different acidic pH’s are compared amongst one another? Joshi et. al. tested this by measuring the rate of deamidation occurring to the Asn & Gln amino acid residues of a glucagon hormone peptide fragment (22-29) across a pH range of 1-3, finding that there appears to be a counterintuitive gradient wherein the more acidic the solution, the faster the rate of deamidation;

Joshi et. al.

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Reconciling both Bhatt et. al. & Joshi et. al.’s findings, it may be safe to assume that, while an acidic pH is preferable as far as peptide stability goes, a pH floor of >3 may present the most promise in keeping a reconstituted peptide in its native, unmodified form. Conveniently, a 0.333% AA solution boasts a pH of 3.

Pain Associated with Injection

In a scoping review on buffers as they pertain to protein formulation, Zbacnik et. al. deduce that both buffer concentration & pH can determine whether an injection will be more likely to produce pain or not, which in turn could jeopardize patient adherence to a dosing protocol. The study authors note that the lower the buffer concentration, and the closer the buffer’s pH to the body’s physiological pH (7.4), the lower the likelihood of injection-associated pain. This assertion on pH, in particular, derives in part from a study carried out by Klement et. al. that found that intravenous injection pain was associated w/ pH levels below 4 & above 11.

Zbacnik et. al.

Klement et. al.

Opting for a reconstitution agent consisting of 0.333% AA as opposed 0.6% would potentially reduce injection-associated pain along both veins: by reducing the buffer concentration by nearly half, and by reducing the acidity by approximately 25% (pH 2.87 -> 3). Of course, bacteriostatic water should be used to draw the IGF-1-peptide-containing AA solution, thereby raising the pH of the injected bolus in its own right, but starting from a pH of 3 as opposed to 2.87 will certainly make the final injection aliquot less acidic.

References

1. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/research-and-disease-areas/cell-and-developmental-biology-research/peptide-stability
2. Fraise AP, Wilkinson MA, Bradley CR, Oppenheim B, Moiemen N. The antibacterial activity and stability of acetic acid. J Hosp Infect. 2013 Aug;84(4):329-31. doi: 10.1016/j.jhin.2013.05.001. Epub 2013 Jun 7. PMID: 23747099.
3. Trzaska WJ, Correia JN, Villegas MT, May RC, Voelz K. pH manipulation as a novel strategy for treating mucormycosis. Antimicrob Agents Chemother. 2015 Nov;59(11):6968-74. doi: 10.1128/AAC.01366-15. Epub 2015 Aug 31. PMID: 26324263; PMCID: PMC4604374.
4. Alphin RL, Johnson KJ, Ladman BS, Benson ER. Inactivation of avian influenza virus using four common chemicals and one detergent. Poult Sci. 2009 Jun;88(6):1181-5. doi: 10.3382/ps.2008-00527. PMID: 19439628.
5. Pianta L, Vinciguerra A, Bertazzoni G, Morello R, Mangiatordi F, Lund VJ, Trimarchi M. Acetic acid disinfection as a potential adjunctive therapy for non-severe COVID-19. Eur Arch Otorhinolaryngol. 2020 Oct;277(10):2921-2924. doi: 10.1007/s00405-020-06067-8. Epub 2020 May 24. PMID: 32449022; PMCID: PMC7245632.
6. https://www.ipsen.com/products/Increlex_Full_Prescribing_Information
7. https://www.cerilliant.com/shopOnline/COA.aspx?itemno=ba0015a9-ce61-4ec3-be57-c298f3db702c&lotno=FN11062013
8. Zbacnik TJ, Holcomb RE, Katayama DS, Murphy BM, Payne RW, Coccaro RC, Evans GJ, Matsuura JE, Henry CS, Manning MC. Role of Buffers in Protein Formulations. J Pharm Sci. 2017 Mar;106(3):713-733. doi: 10.1016/j.xphs.2016.11.014. Epub 2016 Nov 26. PMID: 27894967.
9. Klement W, Arndt JO. Pain on i.v. injection of some anaesthetic agents is evoked by the unphysiological osmolality or pH of their formulations. Br J Anaesth. 1991 Feb;66(2):189-95. doi: 10.1093/bja/66.2.189. PMID: 1817619.
10. Bhatt NP, Patel K, Borchardt RT. Chemical pathways of peptide degradation. I. Deamidation of adrenocorticotropic hormone. Pharm Res. 1990 Jun;7(6):593-9. doi: 10.1023/a:1015862026539. PMID: 2164192.
11. Joshi AB, Kirsch LE. The relative rates of glutamine and asparagine deamidation in glucagon fragment 22-29 under acidic conditions. J Pharm Sci. 2002 Nov;91(11):2331-45. doi: 10.1002/jps.10213. PMID: 12379918.

IGF-1 DES, or more scientifically correct, Des(1-3)IGF-1 (also labeled -3N:IGF-1 in some literature), is a naturally occurring IGF-1 analog, presumably produced post-translationally^1,2,3 via removal of a 3-amino acid sequence (Gly-Pro-Glu) from the N-terminal domain. It’s one of two commonly purchased lyophilized IGF-1 variants, sitting side-by-side w/ IGF-LR3 (LongR³IGF-1) in “research chemical” catalogs, though it’s largely eclipsed by the LR3 variant, probably due to being perceived as being comparatively too short-acting of a compound to get meaningful effects as it pertains to muscular hypertrophy & more. It accordingly is often described as the IGF-1 peptide that is ideal for site-growth, though there is no scientific literature that suggests this to be true. In other words, it’s simply broscience. Indeed, articles & comments written about both IGF LR3 & IGF DES on bodybuilding & biohacking platforms seem to inundated with misinformation. This article will progressively inch its way towards attacking that, beginning with critical background information on Des(1-3)IGF-1, and ending with a more jarring comparison with its more popular cousin, LongR³IGF-1. As a forewarning, many studies referenced throughout this article involve experiments done on mammalian species outside of humans, as no results of any study on metabolism & elimination kinetics in humans following IGF-1 analog administration were ever published, due likely to IGF-1 analogs never having received marketing authorization⁸.

Naturally Occurring Across Several Mammalian Species

While IGF-LR3 tends to steal the spotlight in bodybuilding & bio-hacking societies, it, at the end of the day, is not a naturally occurring IGF-1 analog. It’s a product of laboratory-based synthesis. The same cannot be written of Des(1-3)IGF-1, which has been isolated & purified from human², bovine³ (think cattle), & porcine⁴ (think pigs) tissues & excretions. With all other variables controlled for, preference for an exogenous agent, in this case an IGF-1 analog, should always be lent to substances that the body is familiar with, and according to this principle, the naturally occurring Des(1-3)IGF-1 clearly holds the advantage over the synthetically produced LongR³IGF-1.

Decreased Sequestration by Binding Proteins (IGF-BP’s)

A critical reason why both IGF-1 LR3 & IGF-1 DES exhibit such exaggerated biological activity relative to IGF-1 is due to their apparent incapacity of being bound by the IGF-1-binding proteins (IGF-BP’s) that otherwise serve to sequester free IGF-1 and render them incapable of binding to the target receptors from which the prominent biological effects will precipitate. In the case of Des(1-3)IGF-1, it is specifically the loss of the glutamate (Glu) amino acid in the N-terminal domain that grants it this almost total loss of ability to bind to IGF-BP’s, and both Bagley et. al. & Ballard et. al. conclude that this is one possible explanation for why Des(1-3)IGF-1 exhibits such pronounced biological activity.

Bagley CJ et. al.

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Ballard FJ et. al.

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Enhanced Biological Activity Relative to IGF-1 & IGF-2

In an in vitro study carried out by Francis et. al. on rat L6 myoblasts (muscle progenitor cells, or baby cells, if you will), Des(1-3)IGF-1 was found to be 3.5-fold more active w/ regards the stimulation of DNA synthesis (a surrogate for mitogenic, or mitosis-inducing, activity). Des(1-3)IGF-1 also achieved equivalent inhibition of protein degradation and stimulation of protein synthesis as IGF-1 concentrations that were 8-fold & 8.4-fold greater, respectively, demonstrating its profoundly exaggerated capacity for biological activity at much lower concentrations/doses. The gaps were even more exaggerated when Des(1-3)IGF-1 was compared to IGF-2.

Francis GL et. al.

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Sara et. al., having isolated & purified Des(1-3)IGF-1 in human fetal brain tissue, found again that this truncated version was more potent than either IGF-1 or IGF-2 in stimulating brain DNA synthesis (and thus, proliferation);

Ogasawara et. al., having isolated Des(1-3)IGF-1 from porcine uterus, applied the IGF-1 analog to both human MCF-7 breast cancer cells and mouse Balb/c 3T3 fibroblasts in an in vitro experiment that found that Des(1-3)IGF-1 was 30-100x more potent at stimulating proliferation than IGF-1;

Bioavailability, Half-Life, & the Related

In an in vivo experiment carried out by Ballard et. al that saw intravenous (i.v.) infusions of either IGF-1, IGF-2, or Des(1-3)IGF-1, it was found that Des(1-3)IGF-1 demonstrated rapid clearance from plasma, w/ clearance rates that are 3-4x higher than IGF-1 & IGF-2⁶. This is a result that is consistent with other IGF-1 analogs that bind poorly to IGF-BP’s, leading authors to infer that IGF-BP’s likely enhance the durability (half-life) of IGF-1 in the body. So it’s a double-edged sword as far as the binding proteins go; while they limit the potency of IGF-1 peptides, they concomitantly also amplify their half-lives, and thereby extend their presence in the body. With that written and accounted for, however, in the same experiment by Ballard et. al., i.v. infused Des(1-3)IGF-1 was still recoverable intact (undegraded) in the plasma (the clear, yellowish fluid portion of blood) at 10-19% of initial values 5hrs post-infusion. When reconciling this remnant percentage of active peptide with the exaggerated potency of the peptide (see previous sections of this article), it’s easy to speculate that biological activity could still be notable even 5hrs post-infusion. Unfortunately, this particular study did not test for biological activity, so we can’t infer as much from Ballard et. al.’s setup. However, an in vivo experiment carried out on pigs by Walton et. al. did find that a single intravenous injection of Des(1-3)IGF-1 produced not only a more profound, but also a longer-lasting, metabolic effect (hypoglycemic response) than an injection of IGF-1, IGF-1 LR3, or any other IGF-1 analog;

Now consider that almost all experiments carried out by individuals purchasing IGF-DES or IGF-LR3 will involve either intramuscular (IM) or subcutaneous (SQ) injections, and not an intravenous (i.v.) infusion, meaning that the effective entry & clearance dynamics for most experiments will shift considerably compared to these aforementioned studies, as absorption dynamics of IM or SQ injections are obviously markedly delayed compared to direct intravenous infusion. With that in mind, it becomes easy to speculate that Des(1-3)IGF-1 might exhibit not only a much more profound biological impact, but also a much longer durability in the body than most bodybuilding/biohacking forums & articles will lead you to believe.

Surprisingly Superior Durability, and Prolonged Detectability, Relative to IGF-LR3

I began this article referencing the commonly-espoused belief amongst those in the bodybuilding & biohacking communities that IGF-1 DES is a short-acting analog compared to LR3, and that this reason alone may be the key reason why most individuals devote their attention almost exclusively towards LongR³IGF-1. How fitting, then, to end the article on this particular note. So far, with a reference to an experiment done in pigs by Walton et. al. in particular, it was delineated how Des(1-3)IGF-1 can produce both a more exaggerated & prolonged biological response than LongR³IGF-1, at least when metabolism is in question. To add, a very recent study by Mongongu et. al. (2021) that was designed to delineate ways to optimize detection of IGF-1 analogs for anti-doping purposes found that, while intramuscularly injected Des(1-3)IGF-1 was detectable in its native, undegraded, & unoxidized form through 24hrs post-injection in a rat model, the native form of LongR³IGF-1 was only detectable through the 4hr mark. This discrepancy in duration will undoubtedly come as a surprise to many a reader. In addition to these observations, it was found that LongR³IGF-1 degraded rapidly into a series of metabolites, an outcome that was subsequently replicated in an in vitro experiment that saw the peptides incubated overnight in human blood (human matrices). The study authors suggest that passive proteolysis could be responsible for IGF-LR3’s degradation into these metabolites, probably due to the action of circulating human peptidases that do not require metabolism in cells. We do not know what the biological impacts of IGF-LR3’s metabolites are, and this is cause for concern, if for no other reason than these degradation products simply not possessing the capacity to elicit the mitogenic or anabolic impacts of an intact LongR³IGF-1 peptide. Oppositely, however, Des(1-3)IGF-1 rendered no degradation products in vivo or in vitro. What was detected at 24hrs post-injection, was what was injected originally: the same native form.

Mongongu et. al.

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This is totally at odds with what you’d typically read on bodybuilding/biohacking websites & forums, wherein IGF-LR3 is typically celebrated as a long-acting & durable IGF-1 analog. This discrepancy lends credence to the notion that a typical reader has to take all the information available to them with several grains of salt. All in all, IGF-DES > IGF-LR3 in many more ways than one, and I expect LR3 to eventually be phased out of production over the coming decades as some of the details outlined above become more mainstream.

References

1. Francis GL, Upton FM, Ballard FJ, McNeil KA, Wallace JC. Insulin-like growth factors 1 and 2 in bovine colostrum. Sequences and biological activities compared with those of a potent truncated form. Biochem J. 1988 Apr 1;251(1):95-103. doi: 10.1042/bj2510095. PMID: 3390164; PMCID: PMC1148968.
2. Sara VR, Carlsson-Skwirut C, Andersson C, Hall E, Sjögren B, Holmgren A, Jörnvall H. Characterization of somatomedins from human fetal brain: identification of a variant form of insulin-like growth factor I. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4904-7. doi: 10.1073/pnas.83.13.4904. PMID: 3460078; PMCID: PMC323852.
3. Francis GL, Read LC, Ballard FJ, Bagley CJ, Upton FM, Gravestock PM, Wallace JC. Purification and partial sequence analysis of insulin-like growth factor-1 from bovine colostrum. Biochem J. 1986 Jan 1;233(1):207-13. doi: 10.1042/bj2330207. PMID: 3954725; PMCID: PMC1153005.
4. Ogasawara M, Karey KP, Marquardt H, Sirbasku DA. Identification and purification of truncated insulin-like growth factor I from porcine uterus. Evidence for high biological potency. Biochemistry. 1989 Mar 21;28(6):2710-21. doi: 10.1021/bi00432a052. PMID: 2730884.
5. Bagley CJ, May BL, Szabo L, McNamara PJ, Ross M, Francis GL, Ballard FJ, Wallace JC. A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide. Biochem J. 1989 May 1;259(3):665-71. doi: 10.1042/bj2590665. PMID: 2730580; PMCID: PMC1138570.
6. Ballard FJ, Knowles SE, Walton PE, Edson K, Owens PC, Mohler MA, Ferraiolo BL. Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I in rats. J Endocrinol. 1991 Feb;128(2):197-204. doi: 10.1677/joe.0.1280197. PMID: 2005410.
7. Walton PE, Dunshea FR, Ballard FJ. In vivo actions of IGF analogues with poor affinities for IGFBPs: metabolic and growth effects in pigs of different ages and GH responsiveness. Prog Growth Factor Res. 1995;6(2-4):385-95. doi: 10.1016/0955-2235(95)00007-0. PMID: 8817682.
8. Mongongu C, Coudoré F, Domergue V, Ericsson M, Buisson C, Marchand A. Detection of LongR³ -IGF-I, Des(1-3)-IGF-I, and R³ -IGF-I using immunopurification and high resolution mass spectrometry for antidoping purposes. Drug Test Anal. 2021 Jul;13(7):1256-1269. doi: 10.1002/dta.3016. Epub 2021 Feb 22. PMID: 33587816.

Every juiced-up animal on social media will tell you that the key to muscle hypertrophy, and thus size gainz, is progressive overload, which isn’t inaccurate by any means. The problem is that, for someone who has hit their ceiling in terms of both muscular strength & size, there is no capacity for progressively overloading the muscle without also concomitantly increasing the risk for serious injury (breaking form, doing drop-sets, etc.). And it’s understandable why these influencers would hyperfixate on progressive overload; AAS (Androgenic Anabolic Steroids), through actions on the androgen receptor itself, often increase the capacity of the muscle to overcome resistances. Some of the most androgenic steroids known to man, like Halotestin (8.5x more androgenic than testosterone), increase your strength within mere weeks, even in the absence of size gains. In this circumstance, the androgen receptor-mediated strength increases allow for the progressive overload that can then invite muscle hypertrophy.