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Stroke is the third leading cause of death in the United States


Stroke is the third leading cause of death in the United States and results in substantial health-

care expenditures; the mean lifetime cost resulting from an ischemic stroke is estimated at

$140,048 per patient, and this estimation is higher for people over 45 years. Nationwide in 2010,

the estimated direct and indirect costs of stroke totaled $73.7 billion [1]. Although many clinical

trials have been completed in stroke patients, none of these have demonstrated protective

efficacy except for thrombolysis [2, 3]. In the case of cardiac arrest and resuscitation only

hypothermia has been shown to have clinical utility [4]. In some sense the two therapies that

have been effective thus far clinically have broad targets, and do not only affect a single injury

mechanism. In contrast, of the failed trials, many targeted neuron-specific injury mechanisms

[5]. This may reflect too narrow a view of what is needed for brain preservation. A large body of

work has shown that astrocytes play key roles both in normal and pathological central nervous

system functioning [6]. Astrocytes are the most abundant brain cell type, and in addition to their

multiple important homeostatic roles, they organize the structural architecture of the brain, help

organize communication pathways, and modulate neuronal plasticity (for recent review see

[7, 8]). Thus, astrocytes are now thought to be important potential targets for manipulation.

Ischemic stroke is caused by an interruption of cerebral blood flow that leads to stress, cell death,

and inflammation. Neurons are more susceptible to injury than astrocytes when studied under

some in vitroconditions [9, 10]. Neurons have less endogenous antioxidants and are susceptible

to excito-toxicity [10]. Both normally and after ischemia, astrocytes support neurons by

providing antioxidant protection [11, 12], substrates for neuronal metabolism [13], and glutamate

clearance REF. Although astrocytes are sometimes more resilient than neurons, injury can result

in impaired astrocyte function even when astrocytes do not die. Impaired astrocyte function can

amplify neuronal death [14]. Therefore, many recent efforts have focused on the astrocyte-

neuron interaction and how astrocyte function can be improved after stroke to enhance neuronal

support and survival [10, 15, 16]. A growing body of data demonstrates that astrocytes play a

multifaceted and complex role in the response to ischemia, with potential to both enhance and

impair neuronal survival and regeneration [17]. Many recent studies focus on the astrocyte-

neuron interaction and several investigate ways in which astrocyte function can be improved

after stroke to enhance neuronal survival.

This review provides a brief overview of the pathophysiological events underlying ischemic

brain damage, and considers how these events affect astrocyte-mediated support of neurons. In

addition, we discuss some experimental approaches to enhance the neuronal supportive role of

astrocytes as a novel strategy against stroke. Finally, we explore how these approaches may

eventually be applied in the clinical setting to improve stroke outcome for patients.


2.1. In Vitro Studies

In vitro studies have provided substantial insight into the mechanisms governing the survival of

astrocytes following simulated ischemia. These investigations have shown that astrocytes are

generally more resistant than neurons to oxygen-glucose deprivation (OGD) performed in media

at physiologically normal pH, an in vitro model of ischemia [10, 18]. Most neurons in astrocyte-

neuronal co-cultures will die after 60–90 min of OGD, while astrocyte cultures only suffer a

similar extent of injury after 4–6 hours [9, 18, 19]. Different astrocyte populations exist and

astrocytes isolated from different brain regions such as cortex, striatum, and hippocampus differ

in their sensitivity to OGD [15, 20, 21]. Furthermore, Lukaszevicz and colleagues [22] reported

that protoplasmic astrocytes lose their integrity faster than fibrous astrocytes, which may explain

the regional differences in susceptibility to ischemia between white matter astrocytes which are

fibrous and grey matter astrocytes that are protoplasmic. Although less susceptible to OGD-

induced damaged in vitro studies have highlighted certain elements that are highly toxic to

astrocytes. For example, acidosis has been found to be very effective in killing astrocytes

[23–26], in contrast to neurons, which are protected in acidic conditions [24, 26].

2.2. Focal Cerebral Ischemia

Much of the information about the recovery of astrocytes in vivo has been provided by studies

using immunohistological markers for astrocyte specific proteins, such as glial fibrillary acidic

protein (GFAP) and glutamine synthetase GS; Fig. 1. Using these markers as tools, several

investigations suggest that astrocytes are better preserved than neurons in animal models of

stroke outside the core where all cells die [27–29]. Though neuronal markers are decreased as

soon as 1 hour after MCAO, GFAP expression is preserved over the first 3 hours of reperfusion

after 2 hour MCAO [29] and GS is increased 3 hours following a 3 hour MCAO [28]. At later

reperfusion periods, GFAP increases in the peri-infarct area that later develops into the glial scar

[29–32]. In contrast, Liu and colleagues [33] reported the deterioration of some astrocyte

markers prior to that of neuronal markers. Discrepancy in findings may be due to differences in

detection (i.e., protein vs. mRNA) and injury paradigms.

Fig. (1)

Expression of different astrocytic proteins following stroke. Increased expression of GFAP is a

hallmark of astrocytes activation, as is induction/re-expression of vimentin. Astrocytes normally

express glutamine synthetase (GS) and S100β, genes …

2.3. Forebrain Ischemia

Excitotoxic neuronal injury is a common mechanism in both acute and chronic

neurodegenerative diseases. It has long been appreciated that inhibition of astrocyte glutamate

uptake [34, 35], and more recently inhibition of astrocyte mitochondrial function [36], impairs

neuronal survival from excitotoxic injury. Brief forebrain ischemia is a model of the delayed

hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation, and in part

involves excitotoxicity. Increased generation of reactive oxygen species (ROS) and

mitochondrial dysfunction in CA1 astrocytes contributes to ischemia-induced loss of GLT-1 and

ultimately to delayed death of CA1 neurons [15]. Our studies and those of other laboratories

have demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes, with

loss of glutamate transport activity and immunoreactivity for glutamate transporter 1 (GLT-1),

occurs at early reperfusion times, hours to days before the death of CA1 neurons [15, 37, 38].

The heterogeneous degeneration of astrocytic processes and mitochondria was tightly associated

with the appearance of disseminated selective neuronal necrosis and its maturation after

temporary ischemia [39]. By electronmicroscopy the same investigators [40] found that focal

infarction is exacerbated by temporary microvascular obstruction due to compression by swollen

astrocytic end-feet. However, hypoxia has multiple effects on astrocytes and their ability to

support neuronal viability [41]. For example, hypoxia induces astrocyte-dependent protection of

neurons following hypoxic preconditioning. Yet, hypoxia induces processes in astrocytes that

augment neuronal death in other situations, such as the coincidence of hypoxia with

inflammatory signaling.


The astrocyte response to ischemia has traditionally been viewed as detrimental to recovery, as

the astrocyte-rich glial scar has both physical and chemical inhibitory properties [42, 43]. As

components of the glial scar, astrocytes exhibit hypertrophied, interdigitated processes that form

a physical barrier. Astrocytes produce inhibitory molecules including chondroitin sulfate

proteoglycans (CSPGs) that contribute to chemical inhibition [44, 45]. In the acute setting,

astrocytic gap junctions may remain open following ischemia [46], allowing substances such as

proapoptotic factors to spread through the syncytium, thereby expanding the size of the infarct

[47]. As discussed below, astrocytes can also produce a variety of pro-inflammatory cytokines.

Many studies have shown that decreased astrogliosis often correlates with decreased infarct size.

Nonspecific inhibition of cell proliferation following ischemia using a cyclin kinase inhibitor

decreases astrocyte proliferation and results in improved functional recovery [48]. In addition,

treatment with alpha-melanocyte stimulating hormone [49], cysteinyl leukotriene receptor

antagonist [50], cliostazol [51], and caffeic acid [52] result in reduced infarct size accompanied

by a decrease in astrogliosis. Treadmill exercise [28] and acupuncture [53] are similarly

associated with improved outcome and reduced astrogliosis. Thus, results from several studies

suggest that treatments that decrease infarct size are often accompanied by attenuated astrocyte

response. Despite the frequent association of decreased astrogliosis with improved outcome, it is

difficult to determine cause and effect, since the extent of astrogliosis likely reflects the severity

of the injury, as well as influencing it.

In addition to their role in glial scar formation, astrocytes also respond to ischemia with functions

important for neuroprotection and repair. These include protecting spared tissue from further

damage [14], taking up excess glutamate as discussed above, rebuilding the blood brain barrier

[54, 55], and producing neurotrophic factors [10]. GFAP knockout mice exhibit larger lesions

than their wild-type littermates following focal ischemia [56], and mice lacking both GFAP and

vimentin have impaired astrocyte activation, decreased glutamate uptake abilities, and attenuated

PAI-1 expression after ischemia [57]. Application of astrocyte-conditioned media after transient

MCAO results in decreased infarct volume and regained blood-brain barrier function [58],

suggesting that factors released by astrocytes following ischemia are important for


Although few studies other than the use of animals lacking vimentin and GFAP have specifically

targeted astrocyte activation after ischemia, there is correlational evidence suggesting that

astrogliosis may be beneficial. Environmental enrichment, which results in reduced infarct size

and improved recovery following ischemia, also leads to increased astrocyte proliferation

[59, 60]. After focal ischemia, aged rats exhibit increased tissue damage and increased astrocyte

hypertrophy, but have decreased astrocyte proliferation compared to young rats [61]. Systemic

infusion of bone marrow stromal cells following MCAO increases gliogenesis and decreases

lesion size [62, 63]. In addition, administration of transforming growth factor α (TGFα), a known

mitogen for astrocytes [64], following MCAO leads to reduced infarct size and improved

functional recovery [65]. Furthermore, ischemic preconditioning that produces a neuroprotective

state leads to prolonged astrocyte expression of Hsp27 [66]. Finally, mice lacking connexin 43,

the gap junction connecting astrocyte networks that is needed for proper neurotransmitter and

potassium regulation, have increased infarcts following MCAO [67]. Thus, astrocytes have the

potential to be both detrimental and beneficial following ischemic insult, making them promising

targets for manipulation to improve outcome.



Inflammation plays both detrimental and beneficial roles in brain ischemia, depending upon the

timing and severity of the inflammation. Within minutes after injury, injured neurons in the core

and penumbra of the lesion and glial cells in the core produce pro-inflammatory mediators,

cytokines, and reactive oxygen species, which activate both astrocytes and microglia [68].

Activated astrocytes can produce the proinflammatory cytokines IL-6, TNFα, IL-1α and β,

interferon γ, and others [68–70]. High levels of these cytokines can be detrimental to ischemic

recovery [71–75] by directly inducing apoptosis of neuronal cells and/or increasing toxic nitric

oxide levels [76] and inhibiting neurogenesis [77]. Indeed, inactivation of astrocyte NfκB

signaling, shown to induce astrocyte production of pro-inflammatory cytokines [78], decreases

cytokine production and protects neurons after ischemic injury [79]. Hsp72 overexpression is

associated with lower NfκB activation and lower TNFα [80]. In addition to cytokines, reactive

astrocytes also produce chemokines following ischemia [81]. Chemokines upregulate adhesion

molecules in vascular endothelial cells, resulting in attraction of immune cells, which may

worsen ischemia-induced damage [82]. Overall, some aspects of the local inflammatory response

contribute to secondary injury to potentially viable tissue and lead to apoptotic and necrotic

neuronal cell death hours to days after injury [83], while other aspects are beneficial.

Although the potential benefits of inflammation after stroke have received relatively little

attention so far, indirect evidence suggests that some specific inflammatory reactions are

neuroprotective and neuroregenerative [84–91]. In addition to providing defense against the

invasion of pathogens, inflammation is also involved in clearing damaged tissue, and in

angiogenesis, tissue remodeling, and regeneration [89]. This is probably best studied in wound

healing, which is severely compromised if inflammation is inhibited [89, 91]. There is also

evidence suggesting that specific inflammatory factors can be protective in some circumstances.

IL-6, produced by astrocytes acutely after MCAO [69], is likely neuroprotective early after

ischemia [84]. Interestingly, ischemic preconditioning resulting in protection appears to be

dependent on TLR-4 signaling, and is accompanied by increased TNFα, NFκB, and COX-2

expression [90]. Indeed, in vitro work has shown that administration of TNFα in combination

with Hsp70 results in decreased expression of pro-apoptotic proteins following hypoxia [88].

Thus, it is important to consider these factors, along with timing, when trying to determine the

best strategy to reduce damage and improve recovery and regeneration.


5.1. Antioxidant Production

One hallmark of the cellular response to ischemia is a rapid, dramatic increase in damaging free

radicals, including nitric oxide (NO), superoxide, and peroxynitrite [92]. Nitric oxide synthetase

levels increase as soon as 10 minutes after induction of MCAO [93], followed by NO production

that persists for at least one week after MCAO [94]. Nitric oxide can cause cell death by

inducing the release of cytochrome-c from mitochondria, leading to apoptosis [95]. Nitric oxide

can also induce necrotic death [96]. Furthermore, the production of nitric oxide and other free

radicals can modify oxidative metabolism and impair ATP production [13, 19]. Changes in

mitochondrial properties can further limit oxidative metabolism [97]. Not surprisingly, several

studies have shown that antioxidant treatment enhances neuroprotection and recovery after

stroke [98–101].

The release of glutathione and SOD by astrocytes has been reported and is suggested to play an

important role in maintaining and enhancing neuronal survival, yet they are able to reduce

ascorbate for further neuronal antioxidant defense Fig. (2) [10, 102–106]. Interestingly, neurons

cocultured with astrocytes exhibit higher levels of glutathione compared with neurons cultured

alone [107]. Although astrocytes upregulate SOD after cerebral ischemia [108], they do not

appear to increase levels of glutathione in ischemic conditions [109]. It is unknown whether

ischemia alters astrocytic ascorbate levels, but osmotic swelling from ischemia results in

increased astrocyte release of ascorbate in vitro [110], suggesting that similar mechanisms may

occur in vivo.

Fig. (2)

Mechanisms of astrocyte support of neurons important in stroke. Antioxidant defense includes

release of glutathione and ascorbate. Regulation of extracellular levels of ions and neuro-

transmitters, especially K+ and glutamate, strongly influences neuronal …

Several treatments that attenuate ischemic injury result in increased glutathione levels [111, 112].

SOD converts superoxide into oxygen and hydrogen peroxide. Similar to glutathione, many

treatments that ameliorate stroke damage are accompanied by an increase in SOD [113, 114].

Furthermore, rodents overexpressing SOD1 have significantly smaller injuries after both focal

and global ischemia [115, 116], while mice with decreased SOD1 have larger infarcts [117].

Finally, ascorbate can also reduce oxidative stress [118]. Treatment with dehydroascorbic acid, a

blood-brain-barrier-permeable precursor to ascorbic acid, is protective after MCAO [119].

Dehydroascorbic acid is taken up by astrocytes and released as ascorbic acid [12], a process

increased by propofol [120], a treatment that can be protective after stroke [121]. In summary,

astrocytes are important producers of antioxidants in the normal CNS, and astrocyte production

of these molecules after stroke may enhance neuronal survival and protect astrocyte function.

5.2. Glutamate Regulation

Astrocytes are key players in the regulation of neuro-transmitters in the CNS. Astrocytes make

glutamine, the precursor for the neurotransmitters glutamate and GABA [122] Fig. (2). Astrocyte

production of neurotransmitter precursors is impaired after MCAO, and alterations in neuro-

transmitter levels occur throughout the brain following stroke, possibly contributing to neuronal

death [123, 124].

Astrocytes are primarily responsible for glutamate uptake in the normal brain using the astrocyte

specific glutamate transporters GLAST and GLT-1 (Fig. 2) [125–127], as excess glutamate leads

to cell death via excitotoxicity [128]. Glutamate transporter levels in astrocytes decrease acutely

following global ischemia [38, 129] and neonatal hypoxia-ischemia [130], most likely

exacerbating neuronal death as a result of glutamate-induced excitoxicity. Despite the therapeutic

potential of increasing astrocyte glutamate transport after stroke, few groups have explored this

possibility. Carnosine, shown to be protective after focal ischemia, may partially be effective

because it prevents loss of GLT-1 on astrocytes, resulting in attenuated excitotoxicity [131]. In a

more direct assessment of how post-ischemic astrocyte glutamate transporters contribute to

neuronal survival, our laboratory has shown that upregulation of GLT-1 on astrocytes using

ceftriaxone protects CA1 neurons after global ischemia [129], similar to its effects in focal

cerebral ischemia [132].

5.3. Potassium Uptake and Energy Metabolism

Astrocytes also regulate neuronal activation by extracellular potassium uptake [133] Fig. (2).

Neurons release potassium after activation, and increased extracellular potassium leads to

neuronal hyperexcitability [133], a phenomenon that occurs in ischemic conditions [134]. In

addition to regulating neuronal activation, proper maintenance of ion gradients, such as

potassium, is important in regulating cell volume in both normal and ischemic conditions

[135, 136]. Astrocytes increase potassium transporter activity in response to transient in

vitro ischemia [137]. Due to its effects on both neuronal activity and cell volume, increasing

astrocytic potassium uptake may be a possible therapeutic target for stroke.

Astrocytes are also integral to normal maintenance of neuronal metabolism. When astrocytes

take up extracellular glutamate as a result of neuronal activity, the Na+/ K+-ATPase, along with

AMPA signaling, triggers astrocyte uptake of glucose from the blood, as astrocytic endfeet

contact capillaries [138, 139]. This glucose is then made into lactate, a substrate for neuronal

energy, to further “fuel” active neurons [140] Fig. (2). As mentioned above, astrocytes produce

glutathione. In addition to its antioxidant properties, glutathione is needed for the conversion of

methylglyoxal, a toxic by-product of metabolism, into D-Lactate by glyoxalase 1 [141].

Although the role of astrocyte metabolism is relatively well-established in normal tissue, the

post-ischemic role of astrocyte metabolism maintenance is less clear [142]. After ischemia,

astrocytes upregulate glucose transporters in order to provide energy to stressed/dying neuronal

cells [143,144]. Ethyl pyruvate, a derivative of the energy substrate pyruvate, is neuroprotective

after stroke only when astrocytes are viable, suggesting that astrocytes are necessary for

improvement in post-ischemic energy metabolism [122].



Although few studies have specifically targeted astrocytes for repair after stroke, there is some

evidence that this can be a successful strategy. Recent results indicate that induction of BDNF in

astrocytes by galectin-1 reduces neuronal apoptosis in ischemic boundary zone and improves

functional recovery [145]. In addition, protection by pyruvate against glutamate neurotoxicity is

mediated by astrocytes through a glutathione-dependent mechanism [146]. Our recent study

demonstrated that enhancing astrocyte resistance to ischemic stress by overexpressing protective

proteins or antioxidant enzyme results in improved survival of CA1 neurons following forebrain

ischemia Fig. (3) [16]. Two well-studied protective proteins, heat shock protein 72 (Hsp72) and

mitochondrial SOD, were genetically targeted for expression in astrocytes using the astrocyte-

specific human GFAP promoter. In both cases protection was accompanied by preservation of

the astrocytic glutamate transporter GLT-1, and reduced evidence of oxidative stress in the CA1

region [16]. Similarly, selective overexpression of excitatory amino acid transporter 2 (EAAT2)

in astrocytes enhances neuroprotection from moderate hypoxia-ischemia [147].

Fig. (3)

Targeted over-expression of Hsp72 in astrocytes reduces the vulnerability of CA1 neurons to

forebrain ischemia. Selective overexpression of Hsp72 in astrocytes by expressing it from the

astrocyte specific GFAP promoter was achieved by unilateral stereotaxic …


Many factors have been identified that likely contribute to the failure in translation seen so far

with stroke therapies. Currently, the only approved stroke therapy is thrombolysis induced by

intravenous administration of recombinant tissue plasminogen activator [148]; however, because

of a short therapeutic time window, only a small fraction of patients benefit from this treatment.

Hypothermia is the only accepted acute treatment to reduce brain injury following cardiac arrest

and resuscitation [4]. Thus far many clinical trials have focused on treatments that would likely

be beneficial to neurons, with fewer studies focused on mechanisms that might benefit all cell

types or specifically targeting other cell types, such as astrocytes. Often the consequence of these

treatments on the astrocyte response is not considered. Several examples of past and ongoing

clinical trials are discussed below, with specific attention to how these treatments may alter

astrocyte response or viability.

Several clinical trials have targeted manipulation of the inflammatory response to ischemia, as

stroke patients with systemic inflammation exhibit poorer outcomes [149]. Although anti-

inflammatory therapy decreases infarct size and improves neurological sequelae in experimental

animal models of stroke [150], human trials with anti-neutrophil therapy have not shown a clear

benefit [151, 152]. In addition, recent clinical trials in which anti-CD11/18 antibodies were

administered to human subjects were unsuccessful [153]. Likewise, a double-blinded, placebo-

controlled clinical trial in which anti–ICAM-1 antibody was administered within 6 hours of

stroke symptoms showed disappointing results [151]. In understanding these results it is

important to recall that while experimental stroke is relatively homogeneous concerning size,

territory, and etiology, with more consistent inflammatory response, human stroke is extremely

heterogeneous [154], with different vascular territories and extents of injury. In addition, these

mediators are known to affect many organ systems beyond the central nervous system. Systemic

administration of anti-inflammatory agents may have exacerbated the relative state of

immunocompromise seen in stroke patients, thereby confounding the outcome. Furthermore,

inflammation and astrocyte response are likely closely connected. Although there is little

evidence for a direct relationship between neutrophils and astrocytes, it has been shown that

mice with a blunted inflammatory response exhibit increased loss of GFAP-positive astrocytes

after cortical stab injury [155]. Because astrocytic glial scar formation is important in protection

of spared tissue from further damage [156], it is possible that treatments that drastically attenuate

inflammation lead to a stunted astrocyte response that is deleterious to recovery.

Another drug that has advanced to clinical study is DP-b99, currently in phase III studies for

acute stroke. DP-b99 is a membrane active chelator derivative of the known calcium chelator,

BAPTA spell out [157]. A lipophilic chelator of calcium, zinc and copper ions, DP-b99

sequesters metal ions only within and in to cell membranes. This clinical trial is especially

attractive because sequestration of calcium, zinc, and copper are potentially beneficial not only

to neurons, but also to astrocytes. It has been shown in Alzheimer’s disease that beta amyloid

increases astrocyte calcium influx, which causes decreased glutathione levels [158]. Zinc

chloride is toxic to astrocytes as well as neurons in vitro [159]. Similarly, astrocytes exposed to

neocuprine exhibit increased copper influx and undergo apoptotic cell death [160]. Approaches

that benefit multiple cell types, including astrocytes, are more likely to be successful.

Other current ongoing clinical trials focus on neuroprotective agents for the purpose of aiding

neurological recovery after stroke. Minocycline (Phase I), edavarone (Phase IV), propanolol (a

β-blocker; phase II and III), and more recently arundic acid have been previously shown to be

protective and enhance neuronal survival in stroke [161–165], though by targeting different

mechanisms. Some additional completed and ongoing trials are summarized in Table 1.

Preclinical research needs to consider these clinical results, and assess effects on astrocytes as

well as neurons.

Table 1

Overview of Some Completed and Ongoing Clinical Trials for Stroke

Although anti-inflammatory strategies to diminish ischemic brain injury have failed thus far,

continued elucidation of the complex interactions involved in modulating the inflammatory

response may still enable novel therapeutic approaches that translate successfully into clinical



Traditionally, stroke research has focused on neurons and often ignored effects on glial cells. It is

increasingly evident that glia are vital to both normal CNS functioning and also play important

roles in neuropathological conditions. Although astrocytes form an inhibitory glial scar following

ischemia, they also perform functions necessary for neuronal survival and well-being, such as

maintaining low extracellular glutamate levels and providing antioxidant protection. Because

they have a great many functions, astrocytes are attractive candidates as therapeutic targets. By

striving to shift astrocytes towards a pro-reparative, neuronal-supportive phenotype following

stroke, future clinical therapies may well be more successful in protecting neurons from ischemic

damage and promoting repair.


This work was supported by NIH grants CM49831, N5053898, and NS014543 to RGG.


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Read More

L-Theanine and Caffeine in Combination Affect Human Cognition as Evidenced by Oscillatory alpha- Band Activity and Attention Task Performance1–3

L-Theanine and Caffeine in Combination Affect

Human Cognition as Evidenced by Oscillatory alpha-

Band Activity and Attention Task Performance1–3

1. Simon P. Kelly,

2. Manuel Gomez-Ramirez,

3. Jennifer L. Montesi, and

4. John J. Foxe*

+Author Affiliations

1. Cognitive Neurophysiology Laboratory, Nathan S. Kline Institute for Psychiatric Research,

Program in Cognitive Neuroscience and Schizophrenia, Orangeburg, NY 10962 and Program

in Cognitive Neuroscience, Department of Psychology, City College of the City University of

New York, New York, NY 10031

1. ↵*To whom correspondence should be addressed. E-mail: foxe@nki.rfmh.org.


Next Section


Recent neuropharmacological research has suggested that certain constituents of tea may have modulatory

effects on brain state. The bulk of this research has focused on either L-theanine or caffeine ingested alone

(mostly the latter) and has been limited to behavioral testing, subjective rating, or neurophysiological

assessments during resting. Here, we investigated the effects of both L-theanine and caffeine, ingested

separately or together, on behavioral and electrophysiological indices of tonic (background) and phasic (event-

related) visuospatial attentional deployment. Subjects underwent 4 d of testing, ingesting either placebo, 100

mg of L-theanine, 50 mg of caffeine, or these treatments combined. The task involved cued shifts of attention to

the left or right visual hemifield in anticipation of an imperative stimulus requiring discrimination. In addition to

behavioral measures, we examined overall, tonic attentional focus as well as phasic, cue-dependent

anticipatory attentional biasing, as indexed by scalp-recorded alpha-band (8–14 Hz) activity. We found an

increase in hit rate and target discriminability (d′) for the combined treatment relative to placebo, and an

increase in d′ but not hit rate for caffeine alone, whereas no effects were detected for L-theanine alone.

Electrophysiological results did not show increased differential biasing in phasic alpha across hemifields but

showed lower overall tonic alpha power in the combined treatment, similar to previous findings at a larger

dosage of L-theanine alone. This may signify a more generalized tonic deployment of attentional resources to

the visual modality and may underlie the facilitated behavioral performance on the combined ingestion of these

2 major constituents of tea.

Previous SectionNext Section


In recent years, several potential health benefits of drinking tea (Camellia sinensis) have come to light through

systematic study of the effects of its constituent compounds (1,2). Although anecdotal evidence abounds, the

psychological and neurophysiological effects of tea have received relatively little experimental investigation and

thus remain unclear. Popular claims have centered on generalized state changes such as the reduction of

stress and induction of relaxed wakefulness. Psychopharmacological studies have indeed demonstrated mood

effects that support these claims and have further shown that tea affects elements of cognition (3,4). Although

caffeine (1,3,7-trimethylxanthine) is by far the constituent most studied, with findings of increased alertness and

speeded reaction time (RT)4 predominant (5,6), there exists evidence that caffeine alone cannot fully account

for the positive effects of tea drinking. Tea has been shown to raise skin temperature to a higher level (7), to

increase critical flicker fusion threshold (4), and to reduce physiological stress responses and increase

relaxation ratings (8) when compared with coffee or other control beverages matched for caffeine level.

L-Theanine (γ-glutamylethylamide), a unique amino acid present almost exclusively in the tea plant, has

recently received research interest in the neuroscience community with findings of neuroprotective effects [see

Kakuda (9)] and mood effects indexed both by subjective self-reports (10) and via psychological and

physiological responses to stress (11). Using electroencephalographic (EEG) recordings in humans, Kobayashi

et al. (12) and Juneja et al. (13) reported that activity within the alpha frequency band (8–14 Hz) increased in

reaction to L-theanine ingestion when measured during a state of rest. This was of interest to the attention

community, as the alpha rhythm has long been known to be sensitive to overall attentional states (i.e., intensity

aspects such as arousal) (14) and, further, is involved in the biasing of selective attention (15,16). In

intersensory attention tasks, where the relevant modality is cued ∼1 s before a compound audiovisual target

stimulus, parieto-occipital alpha power in the intervening period is increased for attend-visual trials relative to

attend-auditory trials (15,17). In Gomez-Ramirez et al. (18), this differential effect of cue information on

anticipatory alpha amplitude was found to be larger on ingestion of 250 mg of L-theanine relative to placebo. In

addition, tonic (background) alpha amplitude was relatively decreased for L-theanine, in apparent contradiction

to the findings of Juneja et al. (13). In a follow-up study, we tested whether an analogous alpha-mediated

attention effect seen in visuospatial attention tasks (16,19–22) is also affected by L-theanine ingestion (M.

Gomez-Ramirez, S. P. Kelly, J. L. Montesi, and J. J. Foxe, unpublished results). L-Theanine, at a dosage of

250 mg, was not found to increase the differential effect of attention. However, in a replication of the previous

intersensory attention study (18), overall tonic alpha was greatly reduced on L-theanine.

An immediate question, given this replication, is whether this tonic alpha reduction occurs at lower dosages

of L-theanine, closer to the amount ingested through a typical serving of tea. In the present study, we

administered a lower dosage of 100 mg to address this. Also of critical interest is whether the ingestion of

caffeine, another major component of tea, exerts behavioral and/or neurophysiological effects during such a

demanding visuospatial attention task, when ingested alone or when ingested together with L-theanine. Here

we present data from a 4-d experiment using a balanced repeated-measures design, with subjects receiving

either placebo (P), L-theanine alone (T), caffeine alone (C), or the combination of L-theanine plus caffeine

(T+C) on each day. We assessed effects of treatment with regard to basic behavioral measures of RT and

accuracy [including the so-called discriminability index (d′), which is independent of individual detection criteria],

and in relation to both tonic and phasic attentional processes as indexed by alpha power.

Previous SectionNext Section



Sixteen (5 female) neurologically normal paid volunteers, aged between 21 and 40 y (mean 27.5 y),

participated in the study. All subjects provided written informed consent, and the Institutional Review Board of

the Nathan S. Kline Institute for Psychiatric Research approved the experimental procedures. All subjects

reported normal or corrected-to-normal vision. Four subjects were left-handed. The mean habitual tea

consumption across the subjects was 3.7 cups/wk, and for coffee, 3.8 cups/wk (∼250 mL/cup). Subjects arrived

at the laboratory in the morning between 0900 and 1200 h, having abstained from all caffeinated beverages for

the previous 24 h.


The timing of treatment administration relative to testing was based on published reports of amino acid

concentration and plasma concentration changes over time.L-Theanine concentration has been found to

increase significantly within 1 h after administration in rats, to continue to increase gradually up to 5 h, and to

decrease thereafter, with complete disappearance evident after 24 h (23). Peak plasma caffeine concentration

is reached between 15 and 120 min postingestion in humans, with a variable half-life typically between 2.5 and

4.5 h (5). Accordingly, participants abstained from consuming caffeine for at least 24 h before testing and

began experimental task runs 30 min after ingestion of any given treatment. Subjects underwent 4 d of testing,

ingesting either placebo, 100 mg of L-theanine, 50 mg of caffeine, or these treatments combined. Subjects

were uninformed of the treatment, which was served in 100 mL of water, with the placebo treatment consisting

only of water. Both theanine and caffeine are tasteless in water solution.

Stimuli and task.

Subjects were seated 150 cm from a CRT monitor and were instructed to maintain fixation on a central cross

(white on midgray background) at all times. Each trial began with a centrally presented arrow cue (“S1”) of 100-

ms duration, with equal probability pointing leftward or rightward toward 1 of 2 bilateral locations centered at a

horizontal distance of 4.2° from the fixation cross and 1.2° above the horizontal meridian. Each location was

marked by 4 dots outlining a 2.4° × 2.4° square. The cue consisted of a circle of 1° diameter with an embedded

arrow, designed to minimize any sensory effects related to physical differences between the left and right cues.

The colors of the arrow and circle were red on green for half of the blocks of recording and green on red for the

other half, with the order counterbalanced across subjects and days of testing. Red and green values were

precalibrated for each subject to be approximately isoluminant by flicker photometry. Then, 933 ms after cue

onset, a second imperative stimulus (“S2”) was presented at the left or right marked location (valid or invalid

with respect to cue direction) with equal probability. The S2s (100 ms duration) consisted of either a white × or

+ (0.75° × 0.75°) embedded in a circular array of 8 small circles such that the overall stimulus diameter was

1.95°. The target stimulus was chosen randomly at the beginning of each experimental run of ∼4.5 min, and

thereafter standard and target stimuli were equally likely on each trial. Subjects were instructed to shift their

attention covertly to the location indicated by the cue, to respond by pressing a mouse button with the index

finger of the right hand when a target S2 appeared on that side, and to ignore stimuli appearing on the invalid

side entirely. Trials were separated by a 1633-ms interval. A total of 100 trials were presented per run. Subjects

completed 20 runs on each day of testing.

Data acquisition.

Continuous EEG data, digitized at 512 Hz, were acquired from 164 scalp electrodes and 4 electro-oculographic

(EOG) electrodes with a pass-band of 0.05–100 Hz. Off-line, the data were low-pass filtered up to 45 Hz and

rereferenced to the nasion. Noisy channels, identified by taking the SD of amplitude over the entire run (from

first to last stimulus presented) and checking whether it is >50% greater than that of at least 3 of the 6 closest

surrounding channels, were interpolated. Horizontal EOG data were recorded using 2 electrodes placed at the

outer canthi of the eyes, allowing measurement of eye movements during testing. Based on a calibrated

mapping of EOG amplitude to visual angle, trials were rejected off-line if eye gaze deviated by >0.5° during the

cue-target interval.

Behavioral data analysis.

We employed a d′ as our principal performance metric, taking into account the accuracy of responding on

nontargets as well as targets and controlling for individual differences in detection criteria. The value of d′ was

derived from the hit rate (proportion of all valid targets detected) and false alarm rate (proportion of all valid

nontargets incorrectly responded to), calculated only from trials containing no eye movements or artifacts.

Ceiling effects on hit rate were corrected in the standard way by assuming 0.5 misses, and similarly, a floor

effect of zero false alarms was corrected to 0.5. RT was measured as the time (in milliseconds) from the point

of S2 onset at which the mouse button was correctly pressed in response to valid target trials.

To control for the potential confound of practice effects on the behavioral data, the order of treatments across

the 4 d of testing was fully counterbalanced across subjects. This is a standard procedure and ensures

unbiased comparison across conditions. However, in the case of the present data, the variance in behavioral

measures arising from the day of testing (order effect) far superseded that arising from treatment. Thus, a

normalization of these measures was necessary to remove the variance caused by practice, and this was

carried out by transforming each data point to a z-score with respect to the mean and SD of all scores

measured on that day (d 1, d 2, d 3, d 4). Because the distribution of scores for each day contains an equal

number of data points from each treatment, it cannot result in any bias for treatment but, rather, optimizes

statistical power to test for treatment effects.

Electrophysiological data analysis.

EEG data were epoched from −300 ms before to 1100 ms after cue onset and baseline-corrected relative to

the interval −100 to 0 ms, with an artifact rejection threshold of ±100 μV applied. Mean alpha amplitude was

calculated using the temporal spectral evolution (TSE) technique (15). TSE is carried out simply by filtering

each epoch with a passband of 8–14 Hz, rectifying, then averaging across trials. The averaged TSE waveforms

were then smoothed by averaging data points within a sliding 100-ms window.

The first analysis concerned tonic (background) alpha amplitude, which was found to decrease on L-theanine in

our previous 2 studies (18, M. Gomez-Ramirez, S. P. Kelly, J. L. Montesi, and J. J. Foxe, unpublished results).

Tonic alpha was measured as the integrated TSE amplitude within the baseline period −200 to 0 before the cue

stimulus, regardless of the direction of attentional deployment (i.e., to the left or right hemifield). The dependent

measure was computed as the baseline alpha amplitude averaged across 6 electrodes, chosen on the basis of

the grand-average scalp distribution of alpha amplitude, collapsed across the 4 d.

In a second analysis, we tested lateralized, anticipatory alpha amplitude for effects of attention and possible

interactions with treatment. We normalized alpha amplitude relative to baseline by dividing the TSE amplitude

by the mean amplitude within the baseline interval (−200 to 0) and log-transforming, making the measure

equivalent to a percentage change from baseline. This narrows down the analysis to attention-related

differential activity, independent of tonic effects. The anticipatory alpha dependent measure was computed as

the integrated TSE amplitude over the postcue interval 500 to 900 ms, ending just before the S2. Amplitude

was averaged across 6 electrodes over each hemisphere, determined based on grand-average difference

topographies (cue-left minus cue-right) collapsed across the 4 d.

Statistical methods.

A 4-d balanced repeated-measures design was employed, with subjects receiving 1 of the 4 treatments

(including placebo) on each day in counterbalanced order. SPSS for Windows (version 12.0) was used for all

statistical analyses. Tests were conducted with an α level of 0.05 unless otherwise stated. In the analysis of

behavioral data, we tested specifically for improvements in performance as a result of any of the 3 treatments.

Thus, 1-tailed, paired t-tests (df = 15) were conducted between the placebo condition and each of the 3

treatments for hit rate, RT, and d′ measures. Because 3 t-tests were performed including the same placebo

data, we applied a Bonferroni-corrected α-level of 0.016 here.

To test for effects of tonic alpha amplitude, a 1-way ANOVA was carried out with the factor of treatment having

the levels P, T, C, and T+C. Follow-up protected ttests were then conducted to unpack significant differences

existing between each of the T, C, and T+C conditions and the P condition. Further post hoc paired

comparisons among the 4 treatment conditions were conducted as appropriate through additional t-tests.

To test for effects on pretarget alpha amplitude a 4 × 2 × 2 ANOVA was carried out with factors of treatment (P,

T, C, T+C), attention (cue-left, cue-right), and hemisphere (left, right). To unpack a potential 3-way interaction,

we reduced the alpha cueing effect (typically seen as a hemisphere × attention interaction) to a single measure

by adding the differential over the 2 hemispheres, i.e., subtracting cue-right from cue-left on the left

hemisphere, subtracting cue-left from cue-right on the right hemisphere, and summing these 2 values. Thus

reduced, testing of treatment effects on the alpha cueing effect, as found in the analogous intersensory study of

Gomez-Ramirez et al. (18), could be done via paired t-tests comparing each of the 3 treatments T, C, and T+C

against P.

Previous SectionNext Section


Behavioral performance.

Behavioral performance was significantly improved on the combined treatment (T+C) in terms of hit rate (P <

0.016) and d′ (P < 0.002). There was also a significant improvement in d′ on C compared with P (P < 0.016),

but not in hit rate. There were no significant effects of L-theanine, and no effects of any of the 3 treatments on

RT (Fig. 1).

 Download as PowerPoint Slide


Mean hit rate (proportion of targets detected) (upper panel), mean d′ (middle panel), and mean RT (lower

panel) when subjects ingested placebo (P),L-theanine (T), caffeine (C), or these treatments combined (T+C).

Values are means (n = 16). Asterisks indicate difference from P: *P < 0.05, **P < 0.01).


There was a significant effect of treatment on tonic alpha amplitude (P < 0.02). Follow-up t-tests revealed that

alpha was significantly lower for T+C than P (P < 0.02). P did not differ from either T or C (see Fig. 2). Tonic

alpha differed between T+C and T (P < 0.005) but not between T+C and C.

 Download as PowerPoint Slide


TSE waveforms at midline electrodes from which the baseline tonic alpha measure was derived (upper panel).

Cue-left and cue-right trials are collapsed. Integrated amplitude over the baseline period for each treatment,

with significant difference from placebo marked with an asterisk (lower panel). The electrodes from which tonic

alpha measures were derived are marked on the 168-channel montage.

The typical alpha cueing effect was readily apparent in both the nonnormalized alpha amplitude waveforms and

normalized pretarget measures (Fig. 3) for each treatment day. A strong attention × hemisphere interaction

(P < 0.0005) was found on the pretarget anticipatory alpha measures as expected, reflecting the typically

observed alpha-mediated cueing effect. In addition, there was a significant 3-way interaction among treatment,

attention, and hemisphere (P < 0.05). When we reduced the alpha cueing effect to a single metric as described

above, the effect was smaller on C than P (P < 0.02) but did not differ for the T or T+C conditions.

 Download as PowerPoint Slide


(Upper panel) TSE waveforms over left and right hemispheres, with cue-left (solid) and cue-right (dashed)

superimposed, collapsed across treatment. The overall alpha-mediated spatial cueing effect is highlighted.

Electrode sites for cueing effect measurement are marked on the montage. (Lower panel) Normalized alpha

measures forming the dependent variable in tests for effects of treatment on the alpha cueing effect (P,

placebo; T, L-theanine; C, caffeine; T+C, combined).

Previous SectionNext Section


This study was aimed at extending our knowledge of the effects of compounds contained in tea on the

cognitive function of attention. Testing relatively low-dosage treatments of L-theanine alone (100 mg), caffeine

alone (50 mg), and their combination, we observed an interesting pattern of effects for both behavioral and

electrophysiological measures. Whereas no behavioral effects on hit rate were apparent for either treatment

alone at the low dosages tested here, when both L-theanine and caffeine were ingested together, hit rate

underwent an enhancement of ∼3%. In terms of d′, improvements were seen for both caffeine alone and L-

theanine plus caffeine, the latter having a larger effect size (0.55 vs. 0.42 calculated as Cohen’s d). Given the

absence of any difference in hit rate for caffeine, the d′ effect must result from subjects making fewer false

alarms on caffeine.

Tonic alpha amplitude was not found to decrease significantly on the lower dosage of L-theanine. This indicates

that the effect is dose dependent because a drop was seen in both of our previous studies using a 250-mg

dosage (18, M. Gomez-Ramirez, S. P. Kelly, J. L. Montesi, and J. J. Foxe, unpublished results). There was,

however, a significant decrease in tonic alpha for the combined treatment. That this decrease marks a synergy

between the 2 compounds is suggested by the numerical difference in the alpha decrease caused by L-

theanine with and without caffeine (Fig. 2). That is, it seems unlikely that the greater decrease on the combined

treatment is simply a linear sum of the decreases from each compound alone. Because only single dosages of

each compound were tested, however, a fair degree of caution is appropriate in the interpretation of synergy at

this juncture. This study marks the third finding of decreased alpha as a result of L-theanine ingestion (albeit a

partial cause here) to date, demonstrating the reliability of the effect. At this point, the question of whether it

translates to an improved functional brain state requires serious consideration. Should the finding of a decrease

be received with positive connotations for health and/or mental capabilities?

In the 80 y since the discovery of alpha waves (24), alpha has been measured in almost any experimental

situation and human population, with significant effects abounding, but with a complicated picture and quite

disparate theoretical frameworks arising (25–26). A consistent principle appears to be that stronger alpha infers

positive functioning across individuals (27,28), whereas phasic changes within individuals reflect immediate

stimulus processing and anticipatory enhancement and/or suppression, with a greater retinotopically specific

decrease in alpha being predictive of better detection performance (21). The tonic depression of alpha during

task performance over the day of testing, as observed here, is neither an individual trait nor a phasic event-

related response but a lasting, tonic treatment effect, making it difficult to draw comparisons with such previous

studies. The finding of increased alpha on ingestion of theanine has previously been taken to indicate

increased relaxation without increased drowsiness (13). But this qualification appears tenuous in light of other

observations of treatment-related increased alpha, e.g., during marihuana-induced euphoria (29). Can “good”

and “bad” really be ascribed to increases and decreases in alpha, in whatever direction? Certainly, that this

treatment-related decrease in tonic alpha does not have negative implications is suggested, if not already by

the fact that tea has been keenly, routinely consumed for centuries, by the concomitant facilitation in behavioral

performance found here in terms of both hit rate and d′.

Previous studies have reported a drop in absolute alpha power during resting with eyes open on ingestion of

caffeine at higher dosages, e.g., 200 mg (30) and 400 mg (31). Although alpha amplitude was numerically

lower on 50 mg of caffeine alone here, this did not reach significance (P = 0.18). From this, it is clear that alpha

effects of both L-theanine and caffeine are dose dependent, demonstrating that full characterization of dose-

response functions in future studies is called for.

Evidence of a synergistic relationship between L-theanine and caffeine has been presented in recent

behavioral studies. Parnell et al. (32) reported improved speed and accuracy on an attention-switching task at

60 min and reduced susceptibility to distracting information during a memory task at both 60 and 90 min

following ingestion of a combination of L-theanine and caffeine in the same dosages as used here. Haskell et

al. (33) administered a large battery of cognitive tests before and after consumption of a drink containing either

placebo, 250 mg of L-theanine, 150 mg of caffeine, or their combination. These authors found improvements in

simple and numeric working memory RT, sentence verification accuracy, and alertness ratings for the

combined treatment but not for either treatment alone. Using a similar crossover design but with a greater

dosage of caffeine (250 mg) than L-theanine (200 mg), Rogers et al. (34) found that L-theanine tended to

counteract the caffeine-induced rise in blood pressure but did not interact with caffeine-induced increases in

either alertness or “jitteriness” on state anxiety scales. Although the measures examined in these investigations

and our study are quite distinct in nature, an emerging possibility is that the presence of synergistic effects

closely hinges on dosages. That is, it may be that theanine was not effective in augmenting the caffeine-

induced effects in Rogers et al. (34) because these were present at a saturated level. In the present study,

lower dosages were used, and a significant drop in tonic alpha was observed for L-theanine and caffeine

ingested together but not for either L-theanine or caffeine when ingested alone. However, the absence of a

significant difference between the caffeine-alone and combined treatments calls for caution in making strong

claims of synergy at this point.

Similar to our previous visuospatial attention study (M. Gomez-Ramirez, S. P. Kelly, J. L. Montesi, and J. J.

Foxe, unpublished results), we did not find any change in the alpha differential cueing effect for the L-theanine-

alone treatment. However, it is interesting that the cueing effect was found to be smaller on caffeine alone but

not for the combined treatment. This result was unexpected and thus will bear replication and further

investigation. For now, it appears that within visual space, attentional biasing as indexed by alpha amplitude is

not affected by L-theanine. In contrast, the cued biasing of attention between sensory modalities does appear

to be affected (18). A tentative interpretation of the current pattern of results is thatL-theanine works to enhance

the tonic apportionment of attentional resources to the visual modality and does so to a significant degree when

a large dosage is ingested by itself or in combination with caffeine when a smaller dosage is ingested.

Other articles in this supplement include references (35–44).

Previous SectionNext Section


 ↵1 Published in a supplement to The Journal of Nutrition. Presented at the conference “Fourth International

Scientific Symposium on Tea and Human Health,” held in Washington, DC at the U.S. Department of

Agriculture on September 18, 2007. The conference was organized by the Tea Council of the U.S.A. and was

cosponsored by the American Cancer Society, the American College of Nutrition, the American Medical

Women’s Association, the American Society for Nutrition, and the Linus Pauling Institute. Its contents are solely

the responsibility of the authors and do not necessarily represent the official views of the Tea Council of the

U.S.A. or the cosponsoring organizations. Supplement coordinators for the supplement publication were

Lenore Arab, University of California, Los Angeles, CA and Jeffrey Blumberg, Tufts University, Boston, MA.

Supplement coordinator disclosure: L. Arab and J. Blumberg received honorarium and travel support from the

Tea Council of the U.S.A. for cochairing the Fourth International Scientific Symposium on Tea and Human

Health and for editorial services provided for this supplement publication; they also serve as members of the

Scientific Advisory Panel of the Tea Council of the U.S.A.

 ↵2 Author disclosures: S. P. Kelly, M. Gomez-Ramirez, and J. L. Montesi, no conflicts of interest; J. J. Foxe

received an honorarium and travel support from the Tea Council of the U.S.A. for speaking at the Fourth

International Scientific Symposium on Tea and Human Health and for preparing this manuscript for publication.

 ↵3 Supported by a grant from the Lipton Institute of Tea in association with Unilever Beverages Global

Technology Centre in Colworth House, Sharnbrook, UK.

 ↵4 Abbreviations used: C, caffeine-alone condition; d′, discriminability index; EEG, electroencephalographic;

EOG, electro-oculographic; P, placebo condition; RT, reaction time; T, theanine-alone condition; T+C,

combined condition; TSE, temporal spectral evolution.

Previous Section



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Fu KM, Foxe JJ, Murray MM, Higgins BA, Javitt DC, Schroeder CE. Attention-dependent suppression of

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 CiteULike

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Antioxidant Extends Lifespan


The National Institute on Aging (NIA) announced the final results of testing from three government labs regarding the

patented antioxidant nordihydroguaiaretic acid (NDGA). All three labs agreed that NDGA extended lifespan by a

resounding 12% in mice (1) (see Figure 1).

When some read this astounding news, they were skeptical. With a note of cynicism and doubt in their voices, they

said this report was probably hyperbole and the Federal government is not to be trusted. Yet these same three

government labs had also conducted lifespan studies with much-hyped anti-aging remedies, resveratrol, curcumin,

green tea, oxaloacetic acid and triglyceride oil (2) and found that these five supplements did not extend lifespan in


During the 1980s, researchers extensively tested NDGA in humans, mice and dogs. Results indicated that NDGA

extended lifespan in a variety of mammals. Even the US Patent and Trademark Office approved these results and

granted a patent. This office granted Dr. Richard Lippman a patent for NDGA, as part of a formula developed to slow

aging and extend human lifespan based on his extensive and convincing NDGA research (3).


Background Study of NDGA

Before Dr. Lippman was awarded a US patent on NDGA, several attorneys voiced skepticism. In firm language, they

stated that every law school student knows that two types of patents are never granted: a patent on a perpetual

motion machine and a patent on a fountain-of-youth remedy. Apparently, Dr. Lippman convinced patent examiners

that his clinical human, mice and dog studies of NDGA were sufficient to warrant a patent with claims to retard human

aging. These studies were also sufficient for the drug licensing authorities of Sweden and Italy to grant Dr. Lippman

marketing rights to sell NDGA under the name ‘Aging Control Formula 228’ (ACF228®).

Interestingly, a prominent American businessman, A. Glenn Braswell, had heard Dr. Lippman’s story, but Braswell

doubted that it was sold at the Vatican pharmacy in Rome, Italy. Consequently, he took his wife on a sudden trip to

Rome—and, to his surprise, found that ACF228 was indeed sold at the Vatican pharmacy with the pope’s blessings!


ACF228® Is Based on Extensive Free Radical Research

Today, we know free radicals are not antiwar activists out on bail. But when Dr. Richard Lippman was doing research

in Sweden many years ago, most people thought the term ‘free radicals’ referred to some kind of hippie politics.


No one then knew about these molecular sharks’ devastating effects on the human body and their role in aging.

Indeed, only twenty-five years ago, free radical chemistry and the toxic effects of free radicals on the human body

were unknown to most of the general public and even to many doctors and medical researchers.


Dr. Lippman first learned about the free radical theory of aging as an undergraduate student. When he began doing

graduate research work in cell biology, he and his colleagues held conferences at Pharmacia-Upjohn and the

University of Uppsala to discuss the exciting findings of Professor Denham Harman, whose experimental work at the

University of Nebraska in the 1950s showed that the life spans of mice could be extended 50%

with special antioxidant supplementation. The press and public responded; “So what?”


However, Sweden is well known in science and engineering for its industrial and technical advances. And Lippman

was the leader of a large medical staff that encouraged progressive research.


Raising Funds for Research

Dr. Lippman wanted to take Harman’s work one-step further and explore the relationship between free radicals and

aging. He turned to Professor Sven Brolin, chair of the University of Uppsala’s Department of Medical Cell Biology

and Professor Gunnar Wettermark, chair of the Royal Institute of Technology’s Department of Physical Chemistry, for

assistance in raising funds for research.


Dr. Lippman was successful, receiving significant medical and chemical grants from the Swedish Research Council to

develop antiaging strategies based on Harman’s groundbreaking discovery of the action of free radicals and the role

of radical scavengers (antioxidants) in destroying or inhibiting them. The Swedish Research Council financed years of

Dr. Lippman’s research at the Royal Institute of Technology in Stockholm and at the University of Uppsala,

Scandinavia’s oldest university, which has an anatomy lecture hall built in the 15th century.


Dr. Lippman’s research into the role that free radicals play in the breakdown of the aging body led him to develop one

of the most potent antioxidant combinations yet known, a unique antioxidant cocktail containing NDGA and called


No Typical Scientist

Dr. Lippman’s normal lab attire—jeans, a khaki shirt, and ostrich leather boots—breaks from the conventional notion

of a white-coated scientist. Before his work in antiaging research that made him famous, he ate junk food. Now, a

typical lunch for him is salmon sashimi and salad or bi bim bop with a bowl of miso soup. He even developed his own

recipe for sugar-free, gluten-free, walnut cinnamon pumpkin muffins.


In speaking, Dr. Lippman presents an easy smile and laugh. He may not look like a typical scientist, but his passion

for longevity research is real. His innovative research into free radical pathology helped put antioxidants on the map,

in the dictionary and in the supermarket.


Once funding was in place, Dr. Lippman gathered a team of five prominent Swedish scientists to help him develop

methods for measuring free radicals and biochemical changes related to aging: Professor Agneta Nilsson, a

nutritionist and alternative medical professional with advanced degrees in nursing and teaching; Dr. Ambjörn Ågren,

MD, PhD, who had received numerous awards in the field of emergency medicine; Professor Mathius Uhlén, PhD, a

civil engineer, molecular biologist, and, later, professor and chair of the Royal Institute of Technology’s Department

Molecular Biology; Evald Koitsalu, an engineer and expert in computer hardware and software; and Dr. Kaj

Alverstrand, a psychologist and consultant to Volvo.


With these tremendous financial and personnel resources, Dr. Lippman was able to achieve great leaps in the field of

antiaging. Indeed, Paul Glenn of the Paul Glenn Foundation for Antiaging Research said that Dr. Lippman’s work

was; “light years ahead of everyone else!”


Dr. Lippman’s research resulted in a patent for NDGA and a product that promotes better health and longevity:



Cellular Model—A Better Choice

The research team’s first task was to find a cellular model rather than an animal model to test for life extension, since

the Harman model of waiting for mice to grow old and die was costly and took years of patience before the results

came in.


The Lippman team had access to many different types of living cells in culture, such as human cells of the heart,

brain, liver and central nervous system. In 1980, Lippman invented special probes that would penetrate cell interiors

without harming them. For the first time in the history of cell biology, scientists were able to measure free radicals in

living cells (4). The first probe, carnitinylmaleate luminol (CML), measured superoxide radicals in live human liver

cells. Dr. Lippman and his team went on to test many different combinations of antiaging nutrients.


Developing the formula combinations was a tedious process. Live cells were harvested from biopsies, then separated

and kept metabolically alive in special culture dishes heated to a constant 98°F. The live cells were removed as

needed by the research team and tested for their health by means such as measurements of adenosine triphosphate

(ATP), the power source or ‘gasoline’ of most cell activities.


Then the cell cultures were impregnated with special CML probes and incubated with different mixtures of vitamins

and known antiaging nutrients. Lippman’s team eventually tested 227 different mixtures to find an optimal mixture

with pronounced longevity-promoting characteristics. Mixture number 228 was found to work best, and this and

several other promising mixtures such as 223 were tested further in mice and human volunteers. Now named

ACF228®, the mixture proved successful in extending mice health and life spans, (see Figure 2)


Scientific Community Astounded

The team published its results in more than twenty prominent medical journals. The work astounded the Swedish

scientific community and Lippman was nominated for a Nobel Prize in Medicine in 1996.


Further tests were conducted on hundreds of human volunteers recruited from several Swedish hospitals (3). The

volunteers were tested to establish their normal levels of fatty-acid peroxides, which are free-radical downstream

products, and then were fed varying amounts of ACF228® and other nutrients. Once again, the mixture known as

ACF228® caused normal peroxide levels to decline the fastest. Lippman and his researchers performed other human

tests that indicated ACF228® also had beneficial effects on the skin and sexual function (3).


“We found that the ACF228® formula truly is beneficial,” Dr. Lippman says. “It was especially helpful for middle-aged

and older people; their liver function became like that of teenagers. Often people experience reduced liver function as

they age, especially if they have abused their bodies with heavy

consumption of alcohol and a high sugar diet, causing metabolic syndrome (5). This nutrient mix offers protection

from a multitude of free radicals in the body.”


Ultimately, the ACF228® formula was approved for use by regulatory agencies in both Sweden and Italy and then

patented in the United States.


Indeed, based on these criteria, Dr. Lippman could rest easy. But he isn’t resting. The energetic, youthful-looking

father of three sons and four grandchildren still goes to his lab daily. And what is this Nobel Prize nominee working on

today for the betterment of humankind tomorrow? Dr. Lippman continues his medical research at the behest of

International Antiaging Systems, focusing on improved methods of delivering important vitamins and hormones via

transdermal patches and creams. “Failure to absorb nutrients is a tremendous problem, and 80% of Americans have

problems swallowing pills and capsules,” he says (5).


“The response to ACF228® worldwide has been enormous,” says Dr. Lippman; “that it is indeed gratifying. You know,

we should all be able to live to 120 years and perhaps even beyond. We don’t because of the free radical damage

and declining repair hormones our cellular systems sustain. Our brains shrink, our arteries become hardened and our

liver function declines, mostly because of free radical pathology and damaged endocrine glands. Aging is the ultimate

disease; if ACF228, with its unique blend of natural ingredients can help people to prevent their premature onset,

then I will have lived my life knowing that it has been a success.”



1.       Strong, R et. al., Oct. 2008, Aging Cell, 7(5), pp. 641-650.

2.       Strong, R et. al, Jan 2013, J Gerontology, 68(1), pp. 6-16.

3.       Harman, D., Jul. 1956, J Gerontology, 11(3), pp. 298-300.

4.       Lippman, R, 1987, US Patent No. 4,695,590.

5.       Lippman, R, 1980, Experimental Gerontology, vol. 20, pp. 46-52.

5.    Lippman, R. 2009, Stay 40, Outskirts Press Inc., Boulder, Colorado.

Read More

Anti-Aging Answer for Women’s Health

AAI is the Anti-Aging Answer for Women’s Health

Women’s hormonal web is complicated; finding the right anti-aging serum can also be challenging. The

hormones governing women’s physical and mental function throughout their lives work together to

maintain a balance. The key hormonal players in women, Estrogen, Progesterone, Testosterone/DH EA,

Human Growth Hormone, Thyroid, & Cortisol, begin to play out of tune as their levels decrease with age.

So when hormones decline or become imbalanced, as in Peri Menopause & Menopause, we start to

notice changes in our bones, muscle strength, the elasticity of our skin, and in our energy, mood,

memory, & libido. The majority of women think that Peri Menopause and Menopause cause inconvenient

symptoms: mood swings, fatigue, hot flashes, & low libido among others. What they don’t know is that

these hormonal imbalances raise our risk for serious diseases such as heart disease, Alzheimer’s,

osteoporosis, obesity, depression, stroke, and cancers; especially breast cancer . Therefore, it is crucial

that we prevent and treat the cause of these symptoms as soon as they start to appear in our late 30’s.

Not only our well being and quality of life depends on it, but our health and risk of disease is optimized by

becoming hormonally balanced.

The “Women’s Health Initiative” (WHI 2002) was a major multi-national study that included thousands of

women on synthetic estrogens (Premarin, Birth Control Pills, Nuvaring) and/or on synthetic progesterones

(Provera, Progestins, Birth Control Pellets/Injections). This study had to be stopped abruptly:

unprecedented for any medical study around the world. The reason was that women in the study were

getting a much higher incidence of heart disease, stroke, blood clots, and breast cancer than women in

the general population. At this point, 08/GYN’s around the country took women off their synthetic

hormonal therapy (synthetic estrogens and progesterones) to prevent these problems, even though they

knew women would have to live with bothersome symptoms and their risk for serious illness would be

greatly increased. Bio-Identical or Non-Synthetic hormonal therapy is the only option for women to treat

Peri & Menopausal symptoms, decrease significantly the risk of age-related diseases, and increase well-

being & quality of life. Most doctors are not familiar with the Bio-Identical hormonal therapy, but experts in

Anti-aging Medicine specialize in this field.

Bio-Identical hormones are the answer!

Women’s reproductive life is composed of 3 stages:

1. Pre Menopause – the PMS Years

2. Peri Menopause- the Rollercoaster Years

3. Menopause/Post Menopause-the End of Periods

Pre Menopause-The PMs Years

During their 20’s and 30’s, women experience fairly regular cycles with balanced estrogen and

progesterone production. But increasingly, younger females who are prone to excess stress, crash diets,

and contraceptive use are not ovulating regularly. Anovulatory cycles can lead to symptoms of

hormonal imbalance, severe PMS, and more serious health issues, such as polycystic ovaries,

endometriosis, and infertility.

Peri Menopause – The Rollercoaster Years

Most women don’t know that there is a period of time called Peri Menopause {of 10-15 years) when the

body gets ready to start Menopause, therefore, they do not associate mood and emotional symptoms with

their hormones or menopause. But they should! Beginning in the late 30’s until menopause (average age

of 51), women begin to experience a whole new world of symptoms, as estrogen and progesterone levels

fluctuate dozens of times a day from wavering ovarian function. These symptoms range from classical

menopause symptoms like hot flashes, night sweats, and mood swings to not so typical ones like high

anxiety, foggy thinking, bone loss, weight gain, depression, fatigue, and low libido. These fluctuations in

hormone levels take women on a rollercoaster ride! At this point, the search for symptom relief begins

with Bio-Identical hormones.

Menopause/Post Menopause- The Ends of Periods

The official start of Menopause is 12 months in a row without a period, occurring around age 51. But it is

not uncommon to see symptoms much sooner. Acute/prolonged stress, for example, can negatively

impact ovarian function and can precipitate premature Menopause in vulnerable women as early as their

mid to late 30’s. Menopause can also be surgically induced through hysterectomy, radiation, or

chemotherapy. We think that hormones control only our sexual and reproductive systems, but hormones

actually regulate the entire body. With fewer hormones to go around, their important role in protecting the

health of the brain, bones, muscles, skin, breasts, and heart are diminished.

Supplementation with Bio-Identical hormones is a matter of life or death at this stage.

Common hormone imbalances in Peri Menopause & Menopause:

The right balance of anti-aging serum and hormones is vital to a woman’s health & well being. During Peri

Menopause & Menopause, hormone levels drop and hormone deficiencies take place. Logically, one

would think that hormonal deficiencies should be resolved with hormonal supplementation. Unfortunately,

there are so many hormones at work in a woman’s body that deficiencies in one hormone may trigger

excesses of ether hormones and this is how imbalances take place. (Remember, low levels of hormone

are as harmful to your health as high levels). The following are the most common hormone imbalances in

women and their symptoms:

1. “Estrogen Dominance”–mood swings, migraines, fat gain, low thyroid, breast cancer risk

2. “Low Estrogen & Progesterone”-hot flashes, night sweats, palpitations, foggy thinking

3. “High Testosterone & DHEA”-acne, hair loss, irritability, belly fat, polycystic ovaries

4. “Low Testosterone & DHEA”-decreases bone/muscle mass, energy, libido (sexual desire)

5. “High or Low Cortisol” (Stressed Adrenals): insomnia, anxiety, chronic fatigue, allergies, food

cravings, low immunity

Do you have symptoms of hormonal imbalance?

Mood Swings

Cold Body Temperature

Scalp Hair Loss

Memory Lapses


Sleep Disturbances

Can’t Lose Weight

Thinning Skin

Bone Loss

Uterine Fibroids

Hot Flashes

Sugar or Salty Cravings

Aches & Pains

Decreased Libido

Tender Breasts

Weight Gain (waist &/or hips)

Heart Palpitations

Fibrocystic Breasts



Polycystic Ovaries

Night Sweats

Increased Body /Facial Hair

Vaginal Dryness

Heavy Menses/Bleeding Changes

Foggy Thinking

Water Retention


Foggy Thinking

Troublesome or persistent symptoms during Peri Menopause & Menopause are a sign

of related imbalances that may be complicating the situation and raising disease risks of

osteoporosis, heart disease, breast cancer, obesity, and Alzheimer’s.   Symptoms from

one woman to another are as highly individual as a thumbprint! If you answered yes to 3

or more symptoms in this questionnaire, you may be hormonally imbalanced:

How can I balance my hormones naturally?

Hormone balance and the knowledge that hormones work together with a healthy mind

and body are the keys to optimal health and Peri & Menopausal relief.

1. First, determine your symptoms

2. Then, test ALL of your hormones to detect specific imbalances

3. Find a board certified doctor in Anti-Aging Medicine (specializing in Bio-Identical

hormonal therapy)

4. NEVER use Estrogen alone (even after Hysterectomy)!

5. ALWAYS use Bio-Identical hormones (the exact chemical structure as the hormones

produced by the body and derived from a natural source)

6. STOP all synthetic hormones-Premarin, Provera, Progestins, and birth control pills,

pellets, injections, & intrauterine devices (They ALL contain synthetic hormones)

7. Supplement with Nutriceuticals (pharmaceutical-grade nutritional supplements that

are prescription strength, free of harmful contaminants, and actually absorbed by the

body)-only way to have the available building blocks to regenerate the body’s tissues

and to work properly

8. Detoxify is key for Bio-Identical hormone treatment to work at its best

9. Limit “Xenoestrogens” (substances that mimic estrogen in the body & precipitate

hormonal imbalances)–for example, pesticides, nail polish, fumes,

10. Use “hormone-free” foods and products

11. Maintain ideal weight (fat cells send signals in the body that cause hormonal It is

medically proven that overweight/obesity is a major risk factor for the major killers in

the US: heart disease, stroke, and cancer. Also, breast cancer patients have a

significant increase in fat mass compared to women without cancer)

12. Boost hormones naturally with exercise-not a substitute for hormonal supplementation

with Bio-Identical hormones; exercise makes hormonal therapy more work at its best,

while becoming more stable

13. Minimize stress and use stress-handling techniques like yoga, meditation, prayer,

14. Stop smoking ASAP

15. Eat more fiber

16. Get deep, restful, uninterrupted 7-8 hours of sleep-this is the only way that body

tissues regenerate after being used during the day

Read More

Anti-Aging Answer for Men’s Good Health with Testosterone

Men are exclusively simple, when it comes to a man’s critical hormones, which build a man up,

and causes regeneration of muscles bones and some extremely crucial brain function. These all

lie in a man’s ability to make youthful blood levels of Testosterone. Always remember it is

testosterone that keeps all muscles in the body at their normal size and ability to function in a

youthful manner.

A man produces testosterone when the posterior pituitary’s release of LH which then triggers

the release of HCG which further instructs a man’s testicles to make testosterone. The other

super powerful regenerator of all cells tissues and organs is HGH human growth hormone.

These two hormones kept at youthful blood levels will stop a man from early stage Andropause.

In fact with the correct program of Antioxidants added to the protocol along with the right

energy metabolites and a man never will enter into the debilitating state of advanced

Andropause…. All medical research scientists agree low testosterone is a main cause of male

Andropause….The inability for a man to make youthful blood levels of testosterone. Interesting

that the virtually all male board of American Medical Association (the AMA) does not recognize

the validity of male Andropause….. Ego and false pride changes nothing, when you can no

longer make the 2 critical male hormones at youthful blood levels, the reality is just like female

menopause, men change….and that change leads to old age, decrepitude which is horrific, and

early death.

Testosterone is made in great abundance in young men age 13 to 30. Testosterone is the

combination on the molecular level of Cholesterol, the B vitamins and Oxygen.

However as we age past age 35 there is a large drop off in about 70% of male production of

testosterone according to the national Institute for Health (NHI) If this drop off in production of

testosterone is to severe and too quick, the heart being a muscle can quickly get too small and

not be able to be strong enough to meet cardiac blood stroke volume….the amount of blood

needed by the entire body per beat of your heart. This can lead to a very sudden electrical

storm within the heart muscle causing severe arrhythmias’ followed by death according to the

Harvard ten year medical study on the human heart…. And we are not talking about a few men

dying this way per year, the CDC estimates nearly 150,000 men die each year caused by the

above mentioned severe loss of testosterone…

All men can avoid the above described horror by taking swift action and getting a blood test

done to see what your testosterone levels are….forget about the reference range we look at

what is the OPTIMAL range for testosterone at youthful levels I keep mine at 800 to 950. Most

men over 45 will come in with blood levels of roughly 250 to 475, AAI’s team of

scientist/doctors consider that range to be dangerously low.

So if you have symptoms of:

1. Low Energy, sudden fat built up around your waistline over the last 2 years,

2. Poor sleep

3. Inability to concentrate

4. Lower sex drive

5. And irritability….you are looking at Low Testosterone

6. If you have severe loss of testosterone you could lose hair, have muscle tears, and even

bone fractures…even if you are in your late 40’s and 50’s

Once we here at AAI restore you to youthful levels of testosterone you will have these great


1. Decrease in body fat

2. Increased Muscle tone

3. Stronger muscles and bones

4. Improved mood

5. Higher sex drive

6. Improved sleep

7. Enhanced sense of well being

8. A stronger healthier heart

9. Increased stamina for work, play, and exercise

There is another factor in young men today showing up with low testosterone. The new

generation of pesticides sprayed on crops to give a bigger yields by killing bugs, is only one

molecule different then Estrogen the prime female hormone. Here at AAI we have a protocol

for removing the entire pesticide residues in your body.

Men need certain other aspects to be exceedingly healthy:

1. Exercise

2. Supplements with Nutraceutical(pharmaceutical-grade nutritional supplements that are

prescription strength, free from all contaminants…and fully absorbed by the body

3. Stop Smoking

4. Eat more fiber from ground organic Flax, prevents intestinal cancers.

5. Minimize stress, with yoga, meditation or structured relaxation exercises 20 min. each


6. Limit Xenoestrogens, substances in your diet that may mimic estrogen.

7. You must get 7 to 8 hours of deep restful, uninterrupted sleep each night

8. Allow us at AAI to place you on the most accomplished hormone optimization protocol

with the correct package of energy supplements and antioxidants.

Another incredible aspect of testosterone is that it can prevent type 2 diabetes and it can help

prevent cancer of the prostate according to the current Harvard Chief of Urology. In 1938 the

then, Chief of Urology at Harvard wrote a research paper with over 22 mistakes in it…one of

which was the statement that testosterone can cause cancer. That mistake in his research is still

quoted unto this very day even though 4 years ago the most accomplished urologist in modern

times at Harvard went into the basement of the medical research building…..found the 1938

research paper, and completely demolished it as mistake riddled especially with regard to

testosterone, saying and publishing that testosterone actually prevents prostate cancer

according to all of his research.

You know… that makes so much common sense….the #1 male hormone being testosterone for

a man, would of course be designed by nature to help all aspects of the male body….which

thankfully it does !

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Erectile dysfunction, loss of libido and low sexual frequency increase the risk of cardiovascular

disease in men with low testosterone.

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Whey Protein Facts and Application, Dr. Lawrence Sosna

by: Dr. Lawrence Sosna

The word Protein means “first substance”. Our first protein food was found in our mother’s milk. Milk

is the only food designed specifically to optimally sustain the life of a mammal. In fact, the root word

for nutrition means to suckle.  As a species, we would not have survived if not for the nutrition and

protection mother’s milk offers.

Whey is one of the two protein groups found in milk. It is a liquid complex consisting of a wide range

of proteins. The other protein group is casein, which curds are made from and then processed into


Whey is an original complete protein food and is considered number one for building and

regenerating our bodies and maintaining a strong immune system.  Our entire metabolic process

relies on the intake of complete protein.  We cycle proteins into amino acids constantly.

Even Hippocrates, the Greek physician of the 5th century B.C., the “father of medicine”, knew the

benefits of whey protein. He stated that the body has an inner adaptive or healing power, and that to

strengthen this healing power, he prescribed serum (liquid whey) to his patients. It was true non-

denatured, native whey. It provided full biological activity and numerous health benefits. All

commercial whey proteins available now are derived from extensively processed milk and

incomparable to the vitality in that 2500 year-old prescription.

It is appropriate to review some important definitions of terms used:

Native Protein: The naturally occurring conformation of a protein. Unaltered by heat, chemicals,

enzyme action or processing. (Native is the same structure and proportion as in the original


Denatured: To cause the tertiary structure of (a protein) to unfold, as with heat, alkali, or acid, so

that some of its original properties, especially its biological activity, are diminished or eliminated. (It

means damaged.)

Undenatured: To undamage. (A term that is used without discretion in the industry and is

misleading. It is not possible for a protein to be undenatured.)

Non-denatured: The same structure and proportion as in the original substance with full biological

activity. (Never damaged.)

Presently, the various commercial methods of processing whey do not improve or even maintain the

fragile immune modulating and regenerative components or the biological activity that was originally

in the milk. Most are overly processed and damaged during the manufacturing process.

There are three commercial production methods, which comprise the majority of available whey

proteins. They are isolates (the most popular), ion-exchange and hydrolyzed forms. They are all

ultra-filtered, cross-flow filtered or micro-filtered via elaborate patented methods developed by large

dairies. The milk used in these three methods undergoes major processing that involves high heat

(often multiple times) and drastic acidification of the whey to produce curds for manufacturing

cheese. These steps denature (damage) the proteins. What is then required is extensive filtration to

remove the many denatured proteins in order to produce the highest percentage of protein.

Unfortunately the fragile vital protein components (immunoglobulins, lactoferring, serum albumin,

etc), which determine the biological activity of the protein, are not retained. The terms undenatured

and cold-processed are prevalent with these commercial products, but once a protein is denatured it

is not possible to undenature it.

The key point in regard to the quality and effectiveness of whey is that the full range of biological

activity and proportion of the protein components be preserved in their original native form as nature

provided. Only whey that is minimally processed and maintained can achieve that goal. Additionally,

the health of the milking cows and quality of the milk is the foundation of this type of product.

Non-denatured whey protein has the highest biological value of any protein. It is a complete

protein, unlike soy, and provides all the essential amino acids in the correct balance. The five major

active proteins of whey are lactoferrin, immunoglobulins, bovine serum albumin, alpha-lactalbumin

and beta-lactoglobulin. There are many whey products available; therefore it is highly advisable to

have in writing from the manufacturer, the treatment of the cows and the processing the milk


Covalent Bonded Cysteine (the non-denatured form), is the critical amino acid required for the all-

important intracellular production of the antioxidant glutathione (GSH). Glutathione is our body’s

master antioxidant and is responsible for numerous defense and repair functions and is an effective

anti-aging substance. Glutathione is best utilized when we produce it internally. Cysteine is very

scarce in our modern diet and therefore glutathione production is limited and deficiency is prevalent.

If cysteine undergoes extreme heating or processing, as most commercial whey products do, it is

denatured and converted to cystine. Covalent-bonded cysteine, active peptides, anabolic growth

factors and enzymes are also present in non-denatured whey protein.

The public is now becoming more aware of the value of quality protein and is choosing whey protein

for many good reasons. Whey protein benefits are numerous, and can yield a wide range of

immune-enhancing properties. It also has the ability to act as an antioxidant, antihypertensive, anti-

tumor, antiviral and antibacterial. A number of clinical trials have successfully been performed using

whey as an antimicrobial agent and in the treatment of cancer, HIV, hepatitis B & C, cardiovascular

disease and osteoporosis. It has a major role in red blood cell production, support in chemotherapy

treatment, safe binding and detoxification of heavy metals, wound healing, growth of new muscle,

weight regulation and the support of numerous immune functions. It is used by populations that have

Chronic Fatigue Syndrome (CFS), Fibromyalgia, Hepatitis, Cancer, HIV/AIDS, Respiratory disease,

cognitive disorder from nutritional compromise and for any sports performance improvement.

Dr. Lawrence Sosna

Dr. Lawrence Sosna Graduated first in his class from the Fairfield College of Myopractics and

Naturopathic Medicine. He is a N.D. and has a PhD in Myology with an emphasis in Orthomolecular

Biochemistry. He strictly practices Integrative Medicine – his research field being cellular

regeneration, Anti-Aging and bio-identical comprehensive hormone replacement therapy. Dr. Sosna

lectures on these topics at symposiums all over the world.

Copyright © January 2005

Whey Protein Facts and Applications


Bonang G, Monintja HE, Sujudi, van der Waaij D. Influence of breastmilk on the development of

resistance to intestinal colonization in infants born at the Atma Jaya Hospital, Jakarta. Scand J Infect

Dis 2000;32:189-196.

Bounous G. Whey Protein concentrate and glutathione modulation in cancer treatment, Anticancer

Res. 2000;20:4785-92

Bounous G, Kongshavn PA. Influence of dietary proteins on the immune system of mice. J

Nutr 1982;112:1747-1755.

Bounous G, Gervais F, Amer V, et al. The influence of dietary whey protein on tissue glutathione and

the diseases of aging. Clin Invest Med 1989;12:343-349.

Bowen J, Noakes M, Clifton P. Whey Protein and body fat loss. Asia Pac J Clinical Nut. 2003; 12:S9

Crinnion WJ. Environmental medicine, part 2 – health effects of and protection from ubiquitous

airborne solvent exposure. Altern Med Rev 2000;5:133-143.

Guimont C, Marchall E, Girardet JM, Linden G. Biologically active factors in bovine milk and dairy

byproducts: influence on cell culture. Crit Rev Food Sci Nutr 1997;37:393-410.

Ha E, Zemel MB. Functional properties of whey, whey components, and essential amino acids:

mechanisms underlying health benefits for active people (review). J Nutr Biochem 2003;14:251-258.

Hakkak R, Korourian S, Ronis MJ, et al. Dietary whey protein protects against azoxymethane-

induced colon tumors in male rats. Cancer Epidemiol Biomarkers Prev 2001;10:555-558.

Jones EM, Smart A, Bloomberg G, et al. Lactoferricin, a new antimicrobial peptide. J Appl

Bacteriol 1994;77:208-214.

Kawase M, Hashimoto H, Hosoda M, et al. Effect of administration of fermented milk containing

whey protein concentrate to rats and healthy men on serum lipids and blood pressure. J Dairy

Sci 2000;83:255-263.

Kennedy RS, Konok GP, Bounous G, et al. The use of a whey protein concentrate in the treatment

of patients with metastatic carcinoma: a phase I-II clinical trial study. Anticancer Res 1995;15:2643-


Kimball SR, Jefferson LS. Control of protein synthesis by amino acid availability. Curr Opin Clin Nutr

Metab Care2002;5:63-67.

Lands LC, Grey VL, Smountas AA. Effect of supplementation with a cysteine donor on muscular

performance. J Appl Physiol 1999;87:1381-1385.

Laursen I, Briand P, Lykkesfeldt AE. Serum albumin as a modulator on growth of the human breast

cancer cell line MCF-7. Anticancer Res 1990;10:343-351.

Levay PF, Viljoen M. Lactoferrin: a general review. Haematologica 1995;80:252-267.

Markus CR, Olivier B, de Haan EH. Whey protein rich in alpha-lactalbumin increases the ratio of

plasma tryptophan to the sum of the other large neutral amino acids and improves cognitive

performance in stress-vulnerable subjects. Am J Clin Nutr 2002;75:1051-1056.

Marshall David Jr., O.D., Ph.D. WHEY PROTEIN REPORT – Review of Various Whey Protein.

Current Concepts on Whey Protein Usage.

Micke P, Beeh KM, Buhl R. Effects of longterm supplementation with whey proteins on plasma

glutathione levels of HIV-infected patients. Eur J Nutr 2002;41:12-18.

Sawatzki G, Rich IN. Lactoferrin stimulates colony stimulating factor production in vitro and in

vivo. Blood Cells1989;15:371-385.

Smithers GW, McIntosh GH, Regester GO, et al. Anti-cancer effects of dietary whey

proteins. Proceedings of the Second International Whey Conference 1998;9804:306-309.

Shah NP. Effects of milk-derived bioactives: an overview. Br J Nutr 2000;84:S3-S10. Sundberg J,

Ersson B, Lonnerdal B, Oskarsson A. Protein binding of mercury in milk and plasma from mice and

man – a comparison between methylmercury and inorganic mercury. Toxicology 1999;137:169-184.

Takada Y, Aoe S, Kumegawa M. Whey protein stimulated the proliferation and differentiation of

osteoblastic MC3T3-E1 cells. Biochem Biophys Res Commun 1996;223:445-449.

Tsuda H, Sekine K, Ushida Y, et al. Milk and dairy products in cancer prevention: focus on bovine

lactoferrin. Mutat Res2000;462:227-233.

Watanabe A, Okada K, Shimizu Y, et al. Nutritional therapy of chronic hepatitis by whey protein

(non-heated). J Med2000;31:283-302.

Walzem RL, Dillard CJ, German JB. Whey components: millennia of evolution create functionalities

for mammalian nutrition: what we know and what we may be overlooking. Crit Rev Food Sci

Nutr 2002;42:353-375.

Yamamura J, Aoe S, Toba Y, et al. Milk basic protein (MBP) increases radial bone mineral density in

healthy adult women. Biosci Biotechnol Biochem 2002;66:702-704.

Back to Top

Whey Protein Quality As Compared to Other Available


The quality of dietary proteins is a vital factor in determining what proteins are the most valuable in

terms of how the body assimilates and utilizes the protein as a resource.

To test these ratios in a protein source we begin with an amino acid analysis, a nitrogen analysis,

and then we proceed to the biologic testing. Measuring changes in the protein of the body is a well

accepted evaluative analysis used to determine protein quality, measured as Biologic Value (BV).

This involves the measurement of nitrogen intake from the protein and the output of nitrogen in the

feces and urine. BV is therefore a measurement of the nitrogen absorbed and utilized by the body.

  • Biologic Value (BV) of Dietary Proteins(1)
  • Protein Biologic Value
  • Whey protein 104
  • Egg 100
  • Cow’s Milk 91
  • Beef 80
  • Fish 79
  • Casein 77
  • Soy 74
  • Potato 71
  • Rice 59
  • Wheat 54
  • Beans 49

As this table shows, the animal proteins are high in BV, and are therefore complete proteins(2).

While vegetable proteins are much more incomplete and retain a lower BV rating, due as well to

their lower digestibility(1). With a mixture of these vegetable proteins the effect of a complete protein

can be produced when eaten in sufficient quantity, but this requires a great deal more total protein to

satisfy these requirements.

Whey Protein Concentrates

The benefits (as shown above) of using a whey protein concentrate (WPC) is great according to the

BV of this protein source. It fulfills the body’s amino acid intake beyond any other source of protein

listed above, as well as being a very versatile dietary food. Our Proserum® native whey protein®

concentrate contains all of the essential amino acids for the body as well as providing cysteine and

glutamine. These amino acids are precursors and are necessary for the production of glutathione, a

vital free radical neutralizer in the body.

WPC is defined as a whey protein concentrate containing approximately 80% protein. Proserum® is

a WPC.

1. Renner E. Milk Protein. In: Milk and Dairy Products in Human Nutrition. Munich:

Volkswirtschaftlicher Verlag, 1983

2. Mahan LK, Escott-Stump S. Proteins. In: Krauses Food Nutrition and Diet Therapy, 9th edition,

Philidelphia: WB Saunders; 1996

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Human Growth Hormone (HGH)

Human Growth Hormone (HGH)

Larry Sosna N.D. PhD HHP

It seems like everyone has heard about HGH Human Growth Hormone. But what is it? And is it

safe to take? HGH is 191 Amino Acids in an Exact Molecular Structure. It is NOT a Steroid! Given Correctly at the right dosage it is very Safe and Effective…

If I may be so bold as to talk to you just like we were friends and neighbors, I would be very


You see over 35 years ago, I was just about to die from a serious yet common virus

which normally just shows up on the lip as a sore. Well, that is just what happened AND for

some reason it jumped right on up through my nose, through my sinus and attacked my brain

and spinal cord. All I heard from the doctors was get ready young man, because it seems like

you’re time is up. But guess what? I sure got lucky and did not die like they all said I would. It

was really bad though. I spent 3 years in bed and another 2 years in a wheel chair until I got a

little bit better and one of my friends, also a doctor told me I should take HGH Human Growth

Hormone…and that I might get better. Well, that was the first good news anyone gave me in 5

whole years.

See, think about it like this. Growth Hormone is like a five star general. It’s very smart and just

like a five star general it can and does give orders and commands to everything in our whole

body. Amazing right! So let say you lived in the condo just down the hall on the same floor as

me and you would come and visit me so I would not get so lonely. Because that’s the kind of

friend you are, a real good and decent one.

You noticed each week I was getting a lot better suddenly, and when you came to visit you

wanted to know how in the world did I go from being flat on my back, to moving around and

even walking a bit. Naturally being a friend you had, and maybe still have lots of questions

about how in the world does Growth Hormone make a guy as bad off as me so much better

week after week.

Well, the research doctors explained it very simply by saying that HGH human growth hormone

or just plain GH growth hormone, being the 5 star general, gives orders to the cells in the body

to repair themselves. Growth Hormone can tell any cell at all…say a skin cell, or a nerve cell, or

a heart cell to repair and regenerate itself during deep restful sleep at night. In my situation I

needed lots of repair and regeneration to the nerve cells of my brain and spinal nerves.

It seemed as if I was the only person to survive this type of horrible viral damage to my brain

and spinal nerves they said maybe a few have lived but in a life long coma. So, please listen to

the next part of my journey… because it’s Super Amazing…They tell me that I am going to Italy

to be treated by a woman who won the Nobel Prize in 1985 exactly the time I needed her

because she discovered something very close to growth Hormone which I needed called Nerve

Growth Factor… which when we are in our mother’s womb…Grows all of the billions of Nerves

that become our BRAIN and spinal nerves. I was the first person on this planet to get shots of

nerve growth factor which Dr. Rita Levi Montalcini won the Nobel Prize for and guess what

happened next???

I made a discovery which made me a little famous within Nerve Scientists and Regenerative

Medicine Experts… few that existed back then. What you ask? I discovered that unless a person

with nerve damage or any other long term illness GETS very youthful blood levels of Growth

Hormone to activate the Nerve Growth Factor! Remember Growth Hormone is the Five Star

General, so I could feel the injections of Nerve Growth Factor not making me better… so I did

some basic arithmetic and decided I needed both Nerve growth Factor and Blood levels of

GROWTH HORMONE which would be normal for the average healthy 14 year old young adult.

In 12 months of doing the treatment this way, I went back to Italy 100% healed. No more wheel

chair. The new program had regenerated all of me and repaired all damaged nerves to my

brain and spine. I got better and in the process of needing to do maintenance each week and

each month throughout the years….I became an acknowledged expert in the field of Growth

Hormone Programs, Regeneration Therapies and all the Tissue Growth Factors of the body

starting with Nerve Growth Factor.

As you age, growth hormone declines dramatically. There are new studies that predict that for

every 18 years after age 18…Growth Hormone Declines by 50%. Thus, according to the University

of North Carolina at Chapel Hill….known for being the best at medical studies based on age and

by large groups of people by their age… called demographic medical research studies… We can

see that if you add 18 years to age 18 in just 3 times a person would go from age 18 to age 54

and we see in just 1 more cycle a person is then age 72  and if we go just 1 more cycle a

person becomes age 90… If we follow this very simple method, we see a virtually perfect

predictive model for all age related disease and illness. In this model, aging is an illness in and of

itself. WHY? Because there is simply not enough growth hormone available at or above age 50

to mobilize or put more simply, to turn on the specific nerve growth factor and all the other

tissue growth factors such as muscle growth factor, or skin growth factor, or liver and kidney

growth factor, BONE GROWTH FACTOR…which decline in tandem with growth hormone to be

able to do youthful levels of cell repair and regeneration and thus all the age related issues

including wrinkled skin seen on the face and age related illnesses debilitate folks in time. It is

horrible to see unnecessary Bone Lose and see little old men and women hunched way

over…unable to straighten back up.

So what can we do? We can give much more youthful blood levels of Growth Hormone by

injection, in fact by a very tiny needle so small most folks hardly if ever even feel it. I certainly

do not feel it and I have been giving myself Growth Hormone shots for 25 years.

What else can we do that very few other places can do? We can give our beloved family of

clients all of the specific Tissue Growth Factors including Nerve Growth Factor. We can get

our clients back to youthful levels of both HUMAN GROWTH HORMONE and ALL TISSUE

GROWTH FACTORS so as to be always consistent in producing cell repair and regeneration for

our client’s entire body and not just a few aspects of the body.

I am now age 63 as of Jan. 27 and I have the physiology of a 30 year old and often feel

younger than that.

Advantages to this protocol:

Better Eyesight vision… especially at Night

Increased Cardiac output (stronger more youthful heart muscle)

Enhanced Sex Drive

Loss of FAT around the waist

Increase in lean body muscle

Better metabolism

Much Faster Wound Healing

Hair and Nails Grow Longer Faster (Women Love it)

Improves Daily Energy

Increases Confidence

Emotional ability to Deal with Stress

Much Improved Sleep

Better Orgasms

According to Dr. Daniel Rudman, in New England Journal of Medicine….Age Reversal

And so much MORE

Please come to AAI and see me and the outstanding loving and caring staff….Because you will

become part of our family of beloved clients by phone or in person we live a great part of our

lives to reverse you’re age and allow you to feel incredibly youthful again.

Kindly, Larry Sosna N.D. PhD HHP

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How to Heal from Extreme Stress in American Culture and the Debilitating Effects

Recently we had a class on Digestion and it became apparent that many eating and digestive disorders were a direct result of how we race ahead at break-neck speed. We seem to be always in some kind of obsessive routine. It may be the phone, constant deal making, overuse of the computer so much so that  the time to properly chew our food 32 times before swallowing does not exist. We are the only country to actually confirm Restless Leg Syndrome as an actual medical illness with a CPT code. The same goes for Acid Reflux Disease. In Europe Chronic Reflux is not part of their medical coding because it does not exist. We talked about the fact that inability to digest our food properly has lead to a multi-billion dollar market in medications for Irritable Bowel Syndrome, Leakey Gut Syndrome, Gurd, Constipation, and a host of other digestive disorders that make 73% of our population miserable.
Chronic high level stress weakens all of the human bodies 9 organ systems starting with the Neuroendocrine system. First the Adrenal glands are taxed so severely that Cortisol is always high in the blood…eventually over time millions of people get adrenal burnout and cannot produce this stress fighting hormone at all. When that happens the Adrenal Hypothalamic Pituitary Axis starts to shut down. The more it shuts down the more ill a person becomes until they are both chronically and acutely and severely sick.
The AHP Axis is the most important one in the whole homeostatic bio-feedback loop and thus the next issue at hand is that the entire immune system becomes so down regulated….folks are picked apart by many different types of viruses, bacteria and a whole host of opportunistic micro-pathogens.
According to the American Psychological Association, Chronic High Level STRESS eats away at our emotional adaptive resources. While its hard for me to believe the APA, always great at statistical research says as of 2014, 40% of the American population cannot get a full nights sleep. They report of that 40% 32% have at least 3 nights a week in which they do not sleep at all…. Again almost hard for me to believe.. yet that is the hard reality and if you fall into that 40% you will have 2 times the risk of a heart attack.
This type of stress causes severe emotional turmoil and can lead to drug abuse and binge eating disorders. Emotional Distress can eventually if not interrupted, cause disabling depression or a complete nervous breakdown.
We need to slow down to start to talk about solutions. Taking a walk outside each day is relaxing. We need to avail ourselves of meditation. Many research studies on Transcendental Meditation( super easy to learn and do 20 min. 2 times a day can reduce stress by 60% There is also research showing TM can heal the adrenal glands and start to heal the entire AHP Axis. Slowing down and making better choices, like chewing you’re food 32 times per bite can and does have a major impact in recovery. Eating whole foods, cutting down on sugars, and doing 15 min. of light Yoga will go a long way to heal completely from full stress burnout. The key is to do these healing choices DAILY we must all learn that is NOT what you do once in a while that hurts us its the destructive daily habits that really produces great harm….
To all of our family of friend our dear clients at AAI Rejuvenation, we wish you a very conscious clear choice to achieve a true balance of moderate stress and the joy of allowing us to assist you with gaining perfected states of good health.
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Scientists Have “Reversed” Autism In Mice

A complex spectrum of disorders, it’s highly unlikely that there’s a single cause for autism. That said, a number of genes have been linked with the condition, so can a bit of genetic tweaking help lessen symptoms? Maybe in the future, a new study suggests, as scientists have now managed to reverse some autism-like behaviors by manipulating a single gene in both young and adult mice, even improving brain function in certain areas.

“This suggests that even in the adult brain we have profound plasticity to some degree,” lead researcher Guoping Feng from the Massachusetts Institute of Technology (MIT) said in a statement. “There is more and more evidence showing that some of the defects are indeed reversible, giving hope that we can develop treatment for autistic patients in the future.”

Called Shank3, the gene contains the instructions for a protein found at the connections, or synapses, between nerve cells across which information flows. As a scaffold, it hooks up receptors for chemical messengers (neurotransmitters) with the inner workings of the cell, helping organize the synapse so that cells can respond to incoming signals. It also helps with the formation of little knobbly bits on neurons called dendrites, which receive synaptic messages.

A small percentage of individuals with autism have been found to be missing the Shank3 gene, and a number of mutations within this gene have also been discovered in those on the autistic spectrum. Exactly how these contribute to the condition remains unclear, although earlier work by Feng has contributed to our understanding. Most notably, deleting Shank3 in mice messed up the synapses in a certain brain region called the striatum, reducing the number of dendrites present, and also led to the development of autism-like behaviors such as deficits in social interaction and repetitive actions.

Read the full article herehttp://www.iflscience.com/health-and-medicine/switching-1-gene-adult-mice-reverses-autism-behaviors

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The Hero In All Of Us

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