Blog Arasys Perfector - Xanya Sofra Weiss

Abstract

Chronic low back pain associated with myofascial trigger point activity has been historically refractory to conventional

treatment (Pain Research and Management 7 (2002) 81). In this case series study, an analysis of 22 patients with

chronic low back pain, of 8.8 years average duration, is presented. Following treatment with frequency-specific

microcurrent, a statistically significant 3.8-fold reduction in pain intensity was observed using a visual analog scale. This

outcome was achieved over an average treatment period of 5.6 weeks and a visit frequency of one treatment per week.

When pain chronicity exceeded 5 years, there was a trend toward increasing frequency of treatment required to achieve

the same magnitude of pain relief.

In 90% of these patients, other treatment modalities including drug therapy, chiropractic manipulation, physical therapy,

naturopathic treatment and acupuncture had failed to produce equivalent benefits. The microcurrent treatment was the

single factor contributing the most consistent difference in patient-reported pain relief.

These results support the observation that rigorously designed clinical investigations are warranted.

Arturo Solis1, Maria E. Lara2 and Luis E. Rendon

Melanin is to the animal kingdom like chlorophyll to the vegetal kingdom1.

Melanin collects energy from lower-energy radiation sources, kicks electrons

into excited states, initiating a process that would end up producing chemical

energy, similar to the way in which photosynthesis supplies energy to plants.

However, the precise roles of melanin during this process are unknown. Here we

show that the increase in the electron-transfer properties of melanin is

independent of the energy of the incident photons. We found in controlled in vivo

assays that melanin has the remarkable capability of converting lower-energy

radiation towards a more useful form of energy. Furthermore, we found that

melanin can break up water molecules and giving up energy suggesting an

additional behavior mode for melanin. Our results demonstrate how members of

the melanin family are likely to function as transducers, oxidizing water, pushing

apart water molecules, as well as recruiting back ions into molecules that are

subsequently polarized again. Melanin drives the photon energy of lower-energy

radiation sources by quenching electrons and initiating an ionic event

independently of their relative energy contention. We anticipate our assay to be

a starting point for more sophisticated photoelectrochemical applications. For

example, the individual and combined action of multiple photovoltaic

applications could be tested, including conducting polymers, for example poly-

(phenylenevinylene) (PPV) derivatives or C60 particles. Furthermore, melanin’s

energy conversion ability is a major target of solar energy conversion

development, and an organic-semiconductor way for photoelectrochemical

applications will be relevant for such developments.

Organic active materials electronic circuits, displays, and sensors will enable a

future generation of electronic products that may eventually enter the

mainstream electronic market. The motivation in using organic active materials

come from their ease in tweaking and matching electronic and processing

properties by chemical design and synthesis, low cost processing based on low

temperature processes, mechanical flexibility, and compatibility with flexible

substrates2 . Within these is the family of organic materials, “The Melanins”. For the

sake of simplicity “melanin” will be used in a generic and unqualified sense,

referring primarily to a synthetic, dopa made form of eumelanin. Melanin can be

envisaged as an energy transducer with the properties of an amorphous

semiconductor; it can absorb many different types of energy and convert it in

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another more useful type of energy. In a very enlightening paper, Michael

Grätzel3 looks into the historical background, present status and development

prospects for a new generation of photovoltaic cells, the photoelectrochemical

cells. Until now, the conversion of sunlight to electrical power has been

dominated by solid state junction devices, often made of silicon. But this

dominance we foresee will fall by the emergence of these new “melanincompound”

made photovoltaic cells, based on their here reported new

photoelectrochemical, conducting, polymer type properties. Melanin offers the

prospect of easy fabrication together with other attractive features, such as low

cost. This extra special new property of melanin will result in a huge progress in

fabricating photoelectrochemical cells and will open up whole new vistas of

opportunity. Contrary to expectation, we have proven that some of the made

by us new devices have strikingly high conversion efficiencies, which compete

with those of conventional devices.

Melanin has proven to be an intractable system to study, in part due to their

strong binding with a protein host under in vivo conditions. It is not understood

whether this protein host is a left-over from the bio-synthesis, or whether it is

crucial for the functionality. Strictly speaking, the term “melanin” should

encompass both the chromophore and the associated protein. However, it has

become the norm to use the term to refer only to the chromophore (the

nominally functional part). The vast majority of studies on the natural system

removes (or ignore) the protein and all studies on synthetic melanin involve the

chromophore only. Unfortunately, the chromophores themselves are virtually

insoluble in most common solvents, and have defied systematic attempts at

characterization by standard analytical methods. The predominant strategy in

the field is to study the photochemical and photophysical properties of isolated

natural models or synthetic analogues without recourse to any consistent

structural model.

Dopa-melanin was synthesized from DOPA (dihydroxyphenylalanine) following

the auto oxidizing procedure4. There is a general agreement that melanin are

macromolecules of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-

carboxylic acid (DHICA). The proportion of these two components in the final

macromolecule varies depending upon the type of natural melanin or the

synthetic route. Recently, melanin have attracted the attention of molecular

biophysicists and the soft matter and functional organic materials communities.5

melanin, possesses an intriguing and rather unique set of physicochemical

properties: strong broad band UV and visible absorption; non-radiative

conversion of photo-excited electronic states approaching unity (extremely low

radiative quantum yield)6; powerful anti-oxidant and free radical scavenging

ability7; and probably most bizarrely of all, electrical conductivity and

photoconductivity in the condensed phase8-9. In relation to the last two

properties, it has even been speculated that melanin may be a bio-organic

semiconductors. In figure 1 it is shown the absorbance as a function of

wavelength of a typical synthetic eumelanin aqueous (0.0025%/wt) solution10.

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Our melanin samples have shown very interesting photoelectrochemical

properties. The relation between activity-structure of this polymer was noticed as

the result of our detailed observations in human retina in vivo, while studying the

three main causes of blindness (glaucoma, diabetes, degeneration by age). We

noticed the effect in glioma growth and angiogenesis of melanin presence in

the ocular liquid, the oxygen content increased up to 34 % which did not come

from ATP. We were initially trying to control angiogenesis by studying the

proangiogenic and antiangiogenic factors and it is well known that one of the

most antiangiogenic factors is a high level of oxygen11. Oxygen has a very

important presence, and their quantity an activity is determinant in the evolution

of these diseases. The explanation about their biological activity and presence in

the ocular liquid can not be separated from melanin presence. Because there is

not a clear understanding of how UV and visible radiation interacts with

melanin’s macromolecule to generate properties such as the monotonic broadband

absorbance12. In an attempt to gain new insight into the dynamics of

energy dissipation in melanin under physiological conditions, Forest and Simon13

undertook a wavelength dependent photo acoustic study. They found (as per

radiative quantum yield measurements) that the majority of absorbed energy is

dissipated non-radiatively within a nanosecond of excitation. However, when the

melanin was excited with 264 nm radiation c. 30% of the absorbed energy was

retained for a period longer than a few hundred nanoseconds. This is an

important contribution because it suggests that UV radiation with wavelengths

below 300 nm generates a longer lived excited state with a significant yield,

which in turn may result in excited state photochemistry, which does not occur

under visible light stimulation.

The observations of their biological effects, allow us, to elucidate the protective

effects on human retinal tissue of melanin, undoubtedly due to melanin’s

oxidizing effect on water, liberating oxygen and separating it from protonichydrogen.

Melanin quenches the photons, and with this energy, breaks the

water molecule; absorption of light by this pigment initiates an ionic event and a

reaction that increases the level of oxygen 34 % in surrounding tissues and even

higher in the ocular liquid. This increased energy event in the human eye is the

first step to initiate the vision phenomenon. A great part of the photonic energy is

transformed by melanin into chemical energy by means of hydrogen generation

which acts as an energy carrier, and throughout the action of NADH and FADH,

gives up this energy to the cell, finally the cell uses it to energize some of the

main chemical reactions that enable vision. Without this energy; there is no vision

at all. Melanin in the ocular fluid has an additional characteristic; it supports the

opposite reaction, that is: back binding of hydrogen and oxygen and giving up

water and electricity. Melanin in human eye is for animal kingdom what

chlorophyll is for vegetal kingdom. Both molecules absorb photons, and initiate

an ionic event with large metabolic consequences. The melanin’s macromolecule

solution retains these properties outside the eye for months, even years,

unlike chlorophyll which is very unstable and totally inactive after 20 seconds

outside the leaf. Melanin’s function to other body structures, has shown to be

essential too and its medical applications are huge but out of scope of this

article.

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We claim that photosynthesis exists not only in vegetables, but in animals too.

Meredith14 question – where has all the excess energy gone? – can be

answered even without a satisfactory knowledge of the molecular structure of

the pigment granules, by saying “just set up a couple of electrodes” and you’ll

get melanin’s photoelectrochemical properties, to co-relate the measured

potential to a specific chemical activity.

We proceeded to test the photo electrochemical properties of melanin by

manufacturing a prototype cell (figure 2). We started with a very simple cell were

the electrolyte was a 1.3% solution of melanin in distilled water, cooper and

aluminum electrodes 2.5 cm apart, cooper wires (covered with silicon) where

attach to the electrodes by glued them down, we noticed that any kind of

welding affected the melanin’s behavior. The cell started to give up electricity

just a few minutes after being ensemble.

The output was similar to that of dye-sensitized solar cells, the output was

remarkably stable even under light soaking for more than 10,000 h. see figure 3.

We believe that developing these prototypes could have a significant impact on

the alternative energy sources research.

As Grätzel3 says, the pioneering photoelectric experiments were done with liquid

not solid state devices, the foundation of modern photoelectrochemistry was

laid down by the work of Brattain and Garret15 and subsequently Gerischer16 who

undertook the first detailed electrochemical and studies of the semiconductor–

electrolyte interface. Research on photoelectrochemical cells went through a

frantic period after the oil crisis in 1973, which stimulated a worldwide quest for

alternative energy sources. Within a few years well over a thousand publications

appeared (see ref. 17 for a list). Most of the investigations focused on two types

of cells. The first type is the regenerative cell, which converts light to electric

power leaving no net chemical change behind. Photons of energy exceeding

that of the band gap generate electron–hole pairs, which are separated by the

electric field present in the space-charge layer. The negative charge carriers

move through the bulk of the semiconductor to the current collector and the

external circuit. The positive holes are driven to the surface where they are

scavenged by the reduced form of the redox relay molecule (R), oxidizing it: h+ +

R = Q. The oxidized form Q is reduced back to R by the electrons that re-enter

the cell from the external circuit. Much of the work on regenerative cells has

focused on electron-doped (n-type) II/VI or III/V semiconductors using

electrolytes based on sulphide/polysulphide, vanadium(II)/vanadium(III) or I2/I–

redox couples. Conversion efficiencies of up to 19.6% have been reported for

multijunction regenerative cells18. The second type, photosynthetic cells, operate

on a similar principle except that there are two redox systems: one reacting with

the holes at the surface of the semiconductor electrode and the second

reacting with the electrons entering the counter-electrode. In the example

shown, water is oxidized to oxygen at the semiconductor photo anode and

reduced to hydrogen at the cathode. The overall reaction is the cleavage of

water by sunlight. Titanium dioxide has been the favored semiconductor for

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these studies, following its use by Fujishima and Honda for water photolysis19.

Unfortunately, because of its large band gap, (3–3.2 eV) TiO2 absorbs only the

ultraviolet part of the solar emission and so has low conversion efficiencies.

Numerous attempts to shift the spectral response of TiO2 into the visible, or to

develop alternative oxides affording water cleavage by visible light, have so far

failed. The width of the band gap is the key about the prospects of

photoelectrochemical cells being able to give rise to competitive photovoltaic

devices, as those semiconductors with band gaps narrow enough for efficient

absorption of visible light. The width of the band gap is a measure of the

chemical bond strength. Conducting polymers, for example poly-

(phenylenevinylene) (PPV) derivatives or C60 particles, are attracting great

interest as photovoltaic materials20-25. Bulk donor–acceptor heterojunctions are

formed simply by blending the two organic materials serving as electron donor

(p-type conductor) and electron acceptor (n-type conductor). The advantage

of these new structures over the flat-junction organic solar cells investigated

earlier20 is the interpenetration of the two materials that conduct positive and

negative charge carriers, reducing the size of the individual phase domains to

the nanometers range. This overcomes one of the problems of the first

generation of organic photovoltaic cells: the unfavorable ratio of exciton

diffusion length to optical absorption length. An exciton is a bound electron–hole

pair produced by absorption of light; to be useful, this pair must reach the

junction and there dissociate into two free charge carriers — but excitons

typically diffuse only a few nanometers before recombining. Light is absorbed

(and generates excitons) throughout the composite material. But in the

composite, the distance the exciton has to travel before reaching the interface

is at most a few nanometers, which is commensurate with its diffusion length.

Hence photo-induced charge separation can occur very efficiently. Conversion

efficiency from incident photons to current of over 50% has been achieved with

a blend containing PPV and methanofullerene derivatives21. The overall

conversion efficiency from solar to electric power under full sunlight achieved

with this cell was 2.5%. Although these results are impressive, the performance of

the cell declined rapidly within hours of exposure to sunlight22. In contrast, the

output of dye-sensitized solar cells is remarkably stable even under light soaking

for more than 10,000 h. similar long-term stability will be required for large-scale

application of polymer solar cells.

DISCUSION

The second order structure array must be a graphite type array, that appear to

be composed not of a single chromophore type (whether they be extended

polymeric or smaller oligomeric systems), but rather of ensembles containing a

range of chemically distinct macromolecules (chemical disorder model).

Whatever the situation is this stacked oligomeric ‘proto-molecules’ consisting of

indolequinone units (we claim, they could easily be polyhydroxindole, figure 4),

arranged14 as figure 5 that appears to agree with the early X-ray diffraction

studies of Thathachari and Blois26. Will favor the formation of molecular orbital

arrangement, and we foresee that, this arrangement between layers will

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accommodate the oxygen in such a way with the nitrogen that will generate a

very strong competition between them to produce protons; once a proton is

created the absorbed photon will provide the necessary energy to recombine

the water molecule with the concomitant product of an electron flow.

We should also not ignore the possibility that once the structure-property

problem is properly solved, designing melanin-inspired functional materials

becomes a real possibility for applications in organic sensors and

optoelectronics.

ALEJANDRA ELIZONDO1, JULIA ARAYA1, RAMÓN RODRIGO2, CINZIA SIGNORINI3, CRISTIANA SGHERRI3, MARIO COMPORTI3, JAIME PONIACHIK4 and LUIS A VIDELA2

ABSTRACT

Our aim was to study the influence of weight loss on the fatty acid (FA) composition of liver and erythrocyte phospholipids and oxidative stress status in obese, non-alcoholic, fatty liver disease (NAFLD) patients. Seven obese NAFLD patients who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y were studied immediately and 3 months after surgery. Seven non-obese patients who underwent anti-reflux surgery constituted the control group. Serum F2-isoprostane levels were measured by GS/NICI-MS/MS and FA composition was determined by GC. At the time of surgery, controls and obese patients exhibited a hepatic polyunsaturated fatty acid (PUFA) pattern that correlated with that of erythrocytes. Three months after surgery, NAFLD patients lost 21% of initial body weight; serum F2-isoprostane levels decreased by 76%; total PUFA, long-chain PUFA (LCPUFA), n-3 PUFA, and n-3 LCPUFA increased by 22, 29, 81, and 93%, respectively; n-6/n-3 LCPUFA ratio decreased by 51%; docosahexaenoic acid/docosapentaenoic acid ratio increased by 19-fold; and the n-3 product/precursor ratio (20: 5 + 22: 5 + 22: 6)/18: 3 increased by 164% (p<0.05). It is concluded that weight loss improves the n-3 LCPUFA status of obese patients in association with significant amelioration in the biomarkers of oxidative stress, membrane FA insaturation, and n-3 LCPUFA biosynthesis capacity, thus representing a central therapeutic issue in the improvement of obesity-related metabolic alterations involved in the mechanism of hepatic steatosis.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is becoming a major cause of chronic liver disease in relation to the increasing prevalence of obesity and type-2 diabetes in the general population. In the setting of obesity, NAFLD encompasses a wide disease spectrum including triacylglycerol (TAG) accumulation in hepatocytes (steatosis) and steatosis with inflammation, fibrosis, and cirrhosis (non-alcoholicsteatohepatitis, NASH) (Ángulo, 2006). These hepatic alterations are related to insulin resistance and oxidative stress as crucial pathogenic factors (Videla et al., 2004a; Ángulo, 2006), phenomena that show interdependency (Videla et al., 2006). In particular, hepatic steatosis can be influenced by insulin resistance through induction of a higher peripheral lipolysis with enhanced fluxes of fatty acids (FA) and glycerol to the liver, thus favouring lipogenesis (Saltiel & Khan, 2001). Oxidative stress associated with NAFLD in obese patients represents a nutritional type of phenomenon, resulting from prolonged excess oxidative load (carbohydrates, lipids) and inadequate nutrient supply (dietary antioxidants) favoring pro-oxidant reactions (Sies et al., 2005). Promotion of long-chain polyunsaturated FA (LCPUFA) peroxidation by oxidative stress (Fernández & Videla, 1996; Videla et al., 2004b) may also favor lipogenesis through LCPUFA depletion, particularly those of the n-3 series (Araya et al., 2004). LCPUFA are known to regulate hepatic lipid metabolism by modulating related enzyme expression at the level of gene transcription, mRNA processing and decay, and post-translational protein modifications, directing FA away from TAG storage and into oxidation and secretion (Clarke, 2004).

Depletion of n-3 LCPUFA in NAFLD is related to several factors, in addition to increased peroxidation by oxidative stress (Araya et al., 2004). These include (i) defective dietary intake of n-3 LCPUFA and of their essential precursor a-linolenic acid (18: 3 n-3) (Baylin et al., 2002); evidenced by their lower adipose tissue levels (Araya et al., 2004); (ii) defective desaturation and elongation of PUFA evidenced by the lower liver product/ precursor ratio (20: 5 + 22: 6) n-3/18: 3 n-3; and/or (iii) higher dietary intake of trans FA such as elaidic acid (18: 1 n-9 trans) (Araya et al., 2004), exerting substantial inhibition of hepatic delta-6 desaturase activity (Das, 2005). Recently, the changes in the LCPUFA pattern observed in the liver from obese patients were found significantly correlated with those in erythrocyte phospholipids (Elizondo et al., 2007). These data suggest that erythrocyte FA composition could be a reliable biomarker of the derangements in liver lipid metabolism leading to steatosis in obese patients (Elizondo et al., 2007), a feature that showed major improvements with weight loss induced by bariatric surgery (Dixon et al., 2004; Klein et al., 2006; Mathurin et al., 2006). In view of these considerations, we tested the hypothesis that depletion of n-3 LCPUFA and enhancement in the oxidative stress status of obese NAFLD patients immediately after surgery are ameliorated upon weight loss. For this purpose, LCPUFA composition was determined in liver and erythrocyte phospholipid fractions in relation to the serum levels of F2-isoprostanes as index of oxidative stress, both in obese NAFLD patients at surgery and three months after subtotal gastrectomy.

MATERIALS AND METHODS

Patients and laboratory investigations

Fourteen subjects who received treatment at the Department of Medicine of the Universidad de Chile Clinical Hospital were studied, including seven NAFLD patients [average body mass index (BMI) of 45.4 ± 2.2 kg/m2, age range of 28-56 years], who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y as a therapy for obesity, and seven non-obese patients [BMI of 22.4 ± 0.6 kg/m2, aged between 23 and 60 years] who underwent anti-reflux surgery (control group). The protocol was explained in detail to the subjects, who then gave their written informed consent to participate in the study before any procedure was undertaken. Exclusion criteria included positive hepatitis B or C serology, positive antibodies (antinuclear, anti-mitochondrial, and anti-smooth muscle antibodies), smoking habits or nonsmokers <1 year cessation, and consumption of more than 40 g of ethanol per week. Nutritional and alcohol consumption histories with anthropometric measurements were obtained. Insulin resistance was calculated from the fasting insulin and glucose values by homeostasis model assessment (HOMA) of insulin resistance analysis [fasting insulin (munits/ ml) x fasting glucose (mmol/l)/22.5] (Matthews et al., 1985) [Controls, 2.0 ± 0.2 (n=7) kg/m2; NAFLD patients, 9.6 ± 3.0; P<0.05 assessed by Mann-Whitney U test]. Both plasma lipid levels (total cholesterol, HDL-cholesterol, LDL-cholesterol, and triacylglycerols) and ALT activity in serum were within normal ranges in the studied groups; two NAFLD patients exhibited serum AST levels >40 IU/1 (57-254 IU/1).

The patients were subjected to a diet of 25 kcal/kg body weight (where 1 kcal = 4.184 kJ), with 30% of the energy given as lipids and 15% as proteins, for at least 2 days prior to surgery, and liver tissue of approximately 2 cm3 for histological diagnoses and lipid composition determination were taken during surgery. The samples were fixed in 10% formalin, paraffin embedded, and sections were stained with hematoxylin/eosin and Van Gieson’s stains. Sections of each liver sample were observed in a blinded manner and evaluated for histological alterations by means of previously defined codes (Das & Kar, 2005). Control patients exhibited a normal liver histology, whereas obese patients showed the presence of macro vesicular steatosis. In the latter group, six patients exhibited steatosis alone, and one patient presented steatosis and lobular inflammation with hepatocyte ballooning (steatohepatitis). Considering that changes in the hepatic composition of n-6 and n-3 PUFA is comparable in obese NAFLD patients with steatosis or steatohepatitis (Araya et al., 2004), all obese patients studied were joined in a single group. The Ethics Committee of the Universidad de Chile Clinical Hospital and that of the Faculty of Medicine, Universidad de Chile, approved the study protocol, which was performed in accordance with the Helsinki Declaration II criteria.

Preparation procedure and gas-mass analysis for plasma F2-isoprostane determination

Venous blood drawn from controls and NADLF patients was collected in apyrogenic EDTA-tubes before and three months after surgery in NAFLD patients. Samples were fractioned for determination of either erythrocyte FA composition or serum F2-isoprostane levels, the latter collected in tubes containing 1 mM butylated hydroxytoluene. The samples were stored at -80°C. The preparation of serum samples prior to gas-mass analysis of F2-isoprostanes involved solid-phase extraction on an octadecylsilane (C18) and silica cartridge followed by thin-layer chromatography (TLC), combined with aminopropyl (NH2) cartridge solid-phase extraction (Signorini et al., 2003). The determinations were carried out by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GS/NICI-MS/MS) analysis. Samples (1 ml) were injected into the gas chromatograph in undecane containing N,0-bis-(trimethylsilyl)trifluoroacetamide (BSTFA). The carrier gas was helium, and methane was used as reagent gas at a flow of 1.2 ml/min. The collision energy used was 1.3 eV. The measured ions were m/z 299 and m/z 303 derived from the typical ions (m/z 569 and m/z 573) produced from 15-F2t-IsoP (the most represented isomer) and the tetradeuterated derivative of PGF2a, respectively. The detection limit was 10 pg/ ml (0.028 nM).

Extraction and separation of liver and erythrocyte membrane phospholipids

Liver tissue dissociation was achieved by homogenization in ice-cold chloroform-methanol (2: 1, v/v) containing 0.01% (w/v) butylated hydroxytoluene using an Ultraturrax homogenizer (Janke & Kunkel, Stufen, Germany). Phospholipids from liver were separated by thin-layer chromatography (TLC) aluminum sheets (20 x 20 cm silica gel 60 F-254; Merck, Santiago, Chile), using a solvent system of hexane/diethyl ether /acetic acid (80: 20: 1, by vol.) and phosphatidylcholine as standard (Skipski et al., 1964). After development of the plate, the solvent was allowed to evaporate, and lipid bands were visualized by exposing the plates to Camag UV (250 nm) lamp designed for use in the TLC laboratory. This solvent system separates phospholipids, cholesterol, triacylglycerols, and cholesterol esters in increasing order of relative mobility. Individual lipid zones were scraped from TLC plates and eluted from the silica gel with chloroform/methanol/water (10: 10: 1, by vol.) (Ruiz-Gutiérrez et al., 1992).

Blood samples drawn in vacutainers containing 5% (w/v) EDTA as anticoagulant were centrifuged at 1800 g and 4°C for 15 minutes to separate erythrocytes. Erythrocyte membranes were obtained according to Huertas et al. (1998), and membrane lipids were extracted as described by Bligh and Dyer (1959). Separation of phospholipid fractions was performed as described for liver phospholipids.

Preparation and analysis of fatty acid methyl esters (FAME)

Fatty acids from liver phospholipids and from erythrocyte membranes were methylated. The phospholipids were eluted from silica gel with two 15-ml portions of chloroform/methanol/water (10: 10: 1, by vol). The solvent was evaporated in a nitrogen stream, and 10 mg of tricosaenoic acid (23: 0, internal standard) was added prior to the esterification with 0.2 N sodium-methanol during 30 minutes at 40°C, and then, with H2S04 methanol as described for alkaline methylation. After the sample was cooled, the fatty acid methyl esters (FAME) were extracted with 0.5 ml hexane. FAME of all samples were analyzed by gas-liquid chromatography (GC). A Hewlett-Packard gas chromatograph (model 6890), equipped with a capillary column (50 m x 0.22 mm BPX70; 0.25U QC 0.08 SGE), was employed to separate FAME. The temperature was programmed from 180 to 230°C at 2°C/min with a final hold, separating 12: 0 to 22: 6,n-3. The temperature of both detector and injector was 240°C. Hydrogen was used as carrier gas, at a flow rate of 1.5 ml/min and split ratio of 1: 80. The FAME were identified by comparison of their retention times with those of individual purified standards and quantified using a Hewlett-Packard integrator (HP 3396 Series III) (Araya et al., 2001).

Statistical analysis

Results are expressed as means ± SEM for the number of patients indicated. Statistical analysis of the differences between mean values from control subjects and obese NAFLD patients immediately after surgery was assessed by the nonparametric Mann-Whitney U test, whereas that between patients with NAFLD immediately after surgery and three months post-surgery was performed by Wilcoxon test. The differences were considered statistically significant at p<0.05. To analyze the association between different variables, the Spearman rank order correlation coefficient was used. All statistical analyses were computed using GraphPad Prism™ version 2.0 (GraphPad Software Inc., San Diego, CA,USA).

RESULTS

Obese patients at surgery exhibited average BMI values (Fig. 1A) and serum F2-isoprostane levels (Fig. IB) 103% and 98% higher than those in non-obese controls (p<0.05), respectively. Three months after surgery, obese patients showed 60% and 23% decrease in BMI and serum F2-isoprotane levels over control values (Fig. 1A and IB), thus representing a net 41% and 76% reduction, respectively (p<0.05) (Fig. 1).

Data presented in Table I show significant decreases in the content of 18: 2 n-6, 20: 4 n-6, 20: 5 n-3, 22: 5 n-3, and 22: 6 n-3 in liver phospholipids from obese patients at the time of surgery over control values, with a marked enhancement in that of 22: 5 n-6. The above data represent no significant changes in n-6 PUFA (Fig. 2A) and n-6 LCPUFA (Fig. 2B) in control and NAFLD patients. However, in obese patients, total PUFA and total LCPUFA were 38% lower (Fig. 2A and 2B); n-3 PUFA and n-3 LCPUFA were 59% lower (Fig. 2A and 2B); the n-6/n-3 LCPUFA ratio was 113% higher (Fig. 2C); and the DHA/ DPA n-6 ratio was 93% lower (Fig. 2D) than control values. The product/precursor ratio (20: 5 + 22: 5 + 22: 6) n-3/18: 3 n-3 was 45% lower in the liver of NAFLD patients over control values (controls, 24.6 ±8.2 [n=7]); obese patients immediately after surgery, 13.4 ± 3.3 [n=7]; p<0.05) (from Table I).

The fatty acid composition of erythrocyte phospholipids was comparable in 18: 3 n-3 and 20: 5 n-3 levels in control and NAFLD patients at the time of surgery, whereas 18: 2 n-6 and 22: 5 n-6 were 96% and 369% higher, respectively; and 22: 5 n-3 and 22: 6 n-3 were 74% and 60% lower, respectively (Table II). In agreement with data in liver phospholipids, erythrocyte phospholipids exhibited comparable values of n-6 PUFA (Fig. 3A) and n-6 LCPUFA (Fig. 3B) in control and NAFLD patients. Furthermore, in comparison to control values, in obese patients total PUFA and total LCPUFA were 30% and 35% lower, respectively (Fig. 3A and 3B); n-3 PUFA and n-3 LCPUFA were 56% and 59% lower, respectively (Fig. 3A and 3B); the n-6/n-3 LCPUFA was 144% higher (Fig. 3C); and the DHA/DPA n-6 ratio was 91% lower (Fig. 3D). Association analyses revealed significant correlations in DHA (r=0.67; p=0.0087) and DPA n-6 (r=0.79; p=0.0007) contents between liver and erythrocyte phospholipids from control and NAFLD patients at the time of surgery. Three months after subtotal gastrectomy, NAFLD patients exhibited erythrocyte phospholipid total PUFA and total LCPUFA levels that were 15% lower (Fig. 3A and 3B) and 20% lower n-3 PUFA and n-3 LCPUFA (Fig. 3A and 3B) than controls, thus eliciting net 54% recovery in total PUFA or LCPUFA and 64% recovery in n-3 PUFA or n-3 LCPUFA. In addition, enhancement in erythrocyte phospholipid n-6/n-3 LCPUFA ratio (Fig. 3C) and diminution in that of DHA/DPA n-6 (Fig. 3D) were normalized 3 months after surgery. Compared to control values, the product/precursor ratio (20: 5 + 22: 5 + 22: 6) n-3/18: 3 n-3 was 72% and 26% lower in obese patients at the time of surgery and 3 months after surgery (controls, 32.4 ±3.3 [n=7]); obese patients at the time of surgery, 9.0 ± 2.8 [n=7](p<0.05 versus controls and patients 3 months after surgery); obese patients 3 months after surgery, 23.8 ±6.4 (n=7), respectively, thus representing a 64% recovery (from Table II).

DISCUSSION

Bariatric surgery is the most effective method of achieving long-term weight control for patients with morbid obesity (Angulo, 2006). Data presented indicate that subtotal gastrectomy in obese patients induced (i) significant weight loss, (ii) reduction in the oxidative stress status evidenced by diminution in serum F2-isoprostane levels, and (iii) improvement in the erythrocyte phospholipid LCPUFA pattern, changes that exhibit a direct correlation with those in liver phospholipids.

Obesity-induced changes in LCPUFA pattern consisted in depletion of n-3 LCPUFA, particularly 20: 5 n-3 (EPA), 22: 5 n-3 (DPA), and 22: 6 n-3 (DHA), with enhancement in 22: 5 n-6 (DPA n-6) and n-6/n-3 LCPUFA ratios, and reduction in DHA/DPA n-6 ratios, in agreement with earlier studies (Araya et al., 2004; Elizondo et al., 2007). Improvement in the n-3 LCPUFA status of cellular phospholipids by bariatric surgery is associated with reduction in the oxidative stress status, suggesting lower n-3 LCPUFA peroxidation, considering that n-3 LCPUFA are highly susceptible to free-radical attack (Sevanian & Hochstein, 1985). In agreement with this proposal, weight loss induced by either bariatric surgery (Emery et al., 2003) or dietary sugar restriction (Leclercq et al., 1999) effectively reduces liver CYP2E1 activity, a major free-radical source associated with increased oxidative stress prevailing NAFLD (Lieber, 2004; Videla et al., 2004a; Orellana et al., 2006). In addition, enhancement in the hepatic desaturase/elongation activity needed for n-3 LCPUFA biosynthesis could also play a role in improving n-3 LCPUFA status, as evidenced by the 64% recovery in the product/precursor ratio (20: 5 +22: 5 + 22: 6) n-3/18: 3 n-3 observed 3 months after subtotal gastrectomy. The latter proposal, however, requires an adequate dietary supply of the essential n-3 LCPUFA precursor 18: 3 n-3 to achieve functional relevance (Baylin et al., 2002). Weight loss-related recovery in the cellular n-3 LCPUFA status may ameliorate the obesity-induced changes in hepatic lipid metabolism leading to steatosis (Araya et al., 2004; Videla et al., 2004a), as n-3 LCPUFA suppress lipogenic gene expression and induce that of genes involved in lipid oxidation (Clarke, 2004; Delarue et al., 2004). This view is in agreement with the significant decrease in the amount of hepatic steatosis reported in obese patients at variable times (10 to 36 months) after bariatric surgery (Dixon et al., 2004; Mattar et al., 2005; Clark et al., 2005; Mathurin et al., 2006; Klein et al., 2006; Jaskiewicz et al., 2006).

Cellular depletion of n-3 LCPUFA, particularly DHA, observed in n-3 fatty acid-deficient animals (Connor et al., 1990; Greiner et al., 2003), human infants fed vegetable oil-based formulas (Makrides et al., 1994), or in obese NAFLD patients (Elizondo et al., 2007), is accompanied by increased n-6 PUFA levels, particularly 22: 5 n-6 (DPA n-6), leading to diminished DHA/DPA n-6 ratios. This compensatory mechanism aimed to conserve polyunsaturation of membrane phospholipids (Moriguchi et al., 2001; Leonard et al., 2004) is not fully operative in obesity, as evidenced by the lower levels of total PUFA found in liver and erythrocyte phospholipids from obese patients compared to control values. However, DHA/DPA n-6 ratios in obese patients 3 months after bariatric surgery are comparable to those in control subjects. The latter finding suggests that weight loss effectively recovers polyunsaturation of phospholipids, which may lead to adequate membrane fluidity and improvement of membrane-mediated processes, such as insulin signaling (Lombardo and Chico, 2006), thus lowering the prevalence of the metabolic syndrome (Mattar et al., 2005; Klein et al., 2006; Jaskiewicz et al., 2006).

Collectively, data presented indicate that subtotal gastrectomy-induced weight loss in obese NAFLD patients has beneficial effects manifested by improvement in the n-3 LCPUFA status, associated with the amelioration of biomarkers of oxidative stress, n-3 LCPUFA biosynthetic capacity, and membrane polyunsaturation. These effects occur in patients that have lost 21% of their initial body weight during the first 3 months after surgery, in agreement with the observation that most weight loss induced by bariatric surgery occurs within the first year (Klein et al., 2006; Ángulo, 2006). The early effects on cellular PUFA pattern and systemic oxidative stress status achieved by subtotal gastrectomy and the normalization of the major metabolic abnormalities of NAFLD elicited by other bariatric surgery procedures after longer periods (Klein et al., 2006; Mathurin et al., 2006) point to weight loss as a central therapeutic issue in the improvement or resolution of the obesity-related complications. Moreover, weight loss may be combined with antioxidants, n-3 LCPUFA (Videla et al., 2006), and/or innocuous cytochrome P450 2E1 inhibitors (Lieber, 2004) to minimize alterations in redox status, membrane polyunsaturation, and insulin signaling involved in hepatic steatosis, which otherwise may lead to disease progression.

We describe the association of recurrent complicated febrile convulsions, developmental delay, ataxia, and obesity in three unrelated girls. The three girls, aged 3 to 4 years, were all born to healthy, nonconsanguineous parents and have normal siblings. Their birth weight was appropriate for gestational age. They are not dysmorphic and have normal head circumference. Development is delayed; they all walked with an ataxic gait after the age of 2 years and started speaking at 3 years. Their growth charts are remarkably alike: they initially had a normal growth curve and around 24 months of age started to gain weight excessively. They all continue to suffer from complicated febrile seizures, which started before 12 months of age, and are resistant to prophylactic anticonvulsants. Metabolic evaluation is normal. They have normal magnetic resonance images and electroencephalograms. Fragile X and Prader-Willi syndromes were ruled out. We suggest that this is a new mental retardation syndrome that should be considered in children with recurrent febrile convulsions, developmental delay, and obesity. In a recent study, mutations in the beta4 calcium channel were identified in the mutant epileptic mouse that presents with epilepsy, mental retardation, and ataxia. We hypothesize that a calcium channel gene may be involved in this syndrome.

Obesity is not just a cosmetic problem, but it can lead to a lot of health problems and complications. The health problems associated with obesity are diabetes, heart diseases, arthritis, stroke, liver disease, gall stones etc.

Obesity is because of eating too many calories and not getting enough physical activities to burn those calories. Excess calories are deposited in the body as fat.

Obesity increases the risk of several health problems like high blood pressure, insulin resistance, type 2 diabetes, heart diseases, stroke, gout, gallstones, colon cancer, sleep apnea and non-alcoholic fatty liver disease.

High blood pressure:

Blood vessels carry blood from heart to different organs of the body and back to heart. The blood vessels have thick but elastic walls for proper flow of blood. Decrease in elasticity of blood vessel wall increases pressure on blood passing through these vessels. Obesity decreases elasticity of blood vessels causing increase in blood pressure.

Diabetes in obesity:

Insulin is required for entry of carbohydrate into cells from the blood. The carbohydrate in cell is utilized for energy production by the cells. Excess deposition of fat in the body causes insulin resistance, because of which, insulin cannot perform its function and sugar cannot enter into cells and remain in blood. This leads to diabetes or high blood sugar. High sugar in blood leads to complications in various organs like kidney, eyes, blood vessel, and heart.

Atherosclerosis or fatty deposits in blood vessels:

Obesity is associated with increase in levels of bad cholesterol in blood. Increase cholesterol in blood causes atherosclerosis or deposition of cholesterol on the walls of blood vessels. Atherosclerosis reduces the elasticity of blood vessels, narrows blood vessels and decreases blood flow through these vessels. All these changes lead to increased risk of heart disease and stroke.

Heart diseases:

Coronary arteries are the blood vessels that supply blood to heart muscles. Atherosclerosis or fatty deposits in coronary arteries in obesity decreases blood supply to heart muscles. Decreased oxygen supply and blood flow to heart can cause angina (chest pain) and complete blockage of blood flow to heart can cause heart attack.

Stroke or paralysis:

Atherosclerosis in arteries of brain can reduce blood supply to the brain. This decrease in blood flow can result in stroke or paralysis.

Arthritis:

Obesity and overweight increases the load on the joints such as the knee, hip and lower back, which can cause the breakdown of cartilage in the joint. Cartilage is a cushion like structure in a joint required for smooth movement of joints. Breakdown of cartilage in obesity results in joint pain and stiffness and other features of osteoarthritis.

Gout:

A type of arthritis caused by the accumulation of uric acid crystals in joints. Obesity is associated with increased accumulation of these solid crystal-like masses in joints, which causes inflammation and pain.

Sleep apnea:

Overweight and excess fat around neck causes narrowing of airways and leads to sleep apnea. In sleep apnea, person snores heavily and stops breathing for short periods, which results in frequent awakening at night.

Fatty liver disease:

Obesity increases the risk of developing liver disease called fatty liver disease due to accumulation of fat in liver.

Gallbladder disease and gallstones:

Obesity increases cholesterol deposition in gall bladder, which can lead to formation of gallstones.

So, obesity can lead to a lot of health problems and other complications.

For details on role of nutrients in various diseases, please visit Diet for Disease and for information on management of obesity by blocking carbohydrate absorption, please visit

Carbohydrate in Obesity website.

Abstract

Objective

To determine whether differences in vascular reactivity existed among normal weight, overweight, and obese older men and women, and to examine the association between abdominal fat distribution and vascular reactivity.

Methods

Eighty-seven individuals who were 60 years of age or older (age = 69 ± 7 yrs; mean ± SD) were grouped into normal weight (BMI < 25; n = 30), overweight (BMI ≥ 25 and < 30; n = 28), or obese (BMI ≥ 30; n = 29) categories. Calf blood flow (BF) was assessed by venous occlusion strain-gauge plethysmography at rest and post-occlusive reactive hyperemia.

Results

Post-occlusive reactive hyperemia BF was lower (p = 0.038) in the obese group (5.55 ± 4.67 %/min) than in the normal weight group (8.34 ± 3.89 %/min). Additionally, change in BF from rest to post-occlusion in the obese group (1.93 ± 2.58 %/min) was lower (p = 0.001) than in the normal weight group (5.21 ± 3.59 %/min), as well as the percentage change (75 ± 98 % vs. 202 ± 190 %, p = 0.006, respectively). After adjusting for age, prevalence in hypertension and calf skinfold thickness, change in BF values remained lower (p < 0.05) in obese subjects compared to the normal weight subjects. Lastly, the absolute and percentage change in BF were significantly related to BMI (r = -0.44, p < 0.001, and r = -0.37, p < 0.001, respectively) and to waist circumference (r = -0.36, p = 0.001, and r = -0.32, p = 0.002).

Conclusion

Obesity and abdominal adiposity impair vascular reactivity in older men and women, and these deleterious effects on vascular reactivity are independent of conventional risk factors.

Background

Obesity affects 130 million adults in the United States, and is a major medical concern due to the associated economic and health consequences. Obesity increases the health care costs of older men and women by 33% and 36%, respectively [1,2]. Furthermore, obesity increases the risk of morbidity and mortality, as more than 45% of the total cases of cardiovascular disease and 13% of all deaths occurring in the United States are attributed to obesity [3-7]. Obesity and abdominal fat distribution are associated with endothelial dysfunction [8], which is an initial step in the pathogenesis of atherosclerosis and strongly linked to cardiovascular mortality in the elderly population [9].

Because the vasodilation occurring after a bout of severe hypoxia is dependent on endothelial cell function [10], endothelial function can be assessed by measuring the change in blood flow after vascular occlusion (i.e., vascular reactivity) [11]. In addition to the effect that obesity exerts on the endothelium, it is also associated with hypertension [12], diabetes [13,14], and cardiovascular disease [14-16]. Because these co-morbid conditions impair endothelial function and vascular reactivity [6], it is unclear whether obesity has a direct influence on endothelial function, or whether the co-morbid conditions are the primary mediators.

The purposes of this study were to determine whether differences in vascular reactivity existed among normal weight, overweight, and obese older men and women free of cardiovascular disease, and to examine the association between abdominal fat distribution and vascular reactivity.

Methods

Subjects

Recruitment

A total of 87 individuals between 60 and 89 years of age participated in this investigation. The subjects were recruited from local newspaper advertisements. Prior to investigation, each subject completed a written informed consent. The study was conducted with the approval of the Institutional Review Board at the University of Oklahoma and the University of Oklahoma, Health Sciences Center.

Inclusion and exclusion criteria

To meet the inclusion criteria, subjects were 60 years of age or older, ambulatory, and living independently at home. The exclusion criteria were evaluated by administering a medical history questionnaire and determining ankle/brachial index [17]. Subjects were excluded with the following medical history (or diagnoses): heart or vascular disease, myocardial infarction, cerebrovascular occurrence, peripheral arterial disease (defined by an ankle/brachial index < 0.90 [18]), diabetes, and smoking within the previous year.

Measurements

Demographic information and determination of groups

During a medical history interview, cardiovascular risk factors and co-morbid conditions were assessed. Height and weight were measured using a stadiometer (SECA Corporation; Columbia, MD) and balance scale (SECA Corporation; Columbia, MD), respectively. Body mass index (BMI) was calculated from the height and weight measurements: BMI = weight (kg)/height (m)2. Based on the BMI values, subjects were either categorized as normal weight (BMI < 25), overweight (BMI ≥ 25 and < 30), or obese (BMI ≥ 30) [19]. A nine-site sum of skinfold assessment (the total sum for bicep, tricep, chest, subscapular, midaxillary, iliac, abdominal, thigh and calf skinfold sites), a waist circumference, and a waist-to-hip ratio were also collected for all subjects and used for analysis.

Peripheral blood flow assessment

Calf blood flow was assessed by venous occlusion strain-gauge plethysmography at rest and following a reactive hyperemic test as previously described [17]. A mercury-in-rubber strain gauge was placed around the calf at the maximal circumference, and blood pressure cuffs were placed around the thigh and ankle. Following 10 minutes of supine rest, the measurement of blood flow was isolated in the calf by inflating the thigh cuff to a venous occlusion pressure of 50 mmHg, and by temporarily occluding arterial blood flow to the foot by inflating the ankle cuff to 200 mmHg. The ankle and thigh cuffs were deflated immediately after the calf blood flow measurement was obtained. The reactive hyperemic test was then performed while patients were in the supine position by inflating a thigh blood pressure cuff to inducing arterial occlusion for three minutes. The thigh blood pressure cuff was inflated to the highest value of either 200 mmHg or 50 mmHg above systolic blood pressure. Post-occlusive reactive hyperemic calf blood flow measurements were obtained within 15 seconds following arterial occlusion. We previously demonstrated that the test-retest intraclass reliability coefficient was R = 0.86 for calf blood flow [20].

Statistical analysis

Descriptive statistics were computed for the physical characteristics and measured variables. Analysis of variance (ANOVA) was used to assess differences among the normal weight, overweight, and obese groups for continuous variables, and chi-square tests were used to assess differences among the three groups for categorical variables. A post hoc analysis with a Bonferroni correction was done to identify groups that were significantly different from each other. Analysis of covariance (ANCOVA) was used to assess group differences in calf blood flow adjusting for differences in physical and clinical characteristics. Pearson correlation coefficients were also calculated to assess the relationships among vascular reactivity, BMI, waist circumference, and waist-to-hip ratio. All values are reported as the mean ± standard deviation (SD). The level of statistical significance for the study was set at p ≤ 0.05. All statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS, v. 11.5, Chicago, IL) software.

Results

The physical and clinical characteristics of the subjects in each weight group are shown in Table 1. Group differences (p ≤ 0.05) were found for age, weight, BMI, waist circumference, waist-to-hip ratio, calf skinfold, sum of skinfolds and hypertension. Weight, BMI, and waist circumference were different (p < 0.017) for each pair-wise group comparison, with the normal weight group having the lowest values, the overweight group having intermediate values, and the obese group having the highest values. Additionally, the waist-to-hip ratio was lowest in the normal weight group (p < 0.017), and the sum of skinfolds was greatest in the obese group (p < 0.017).

Table 1. Physical and clinical characteristics of normal weight, overweight, and obese subjects. The p value represents the significance among the three weight groups.

The calf blood flow measurements of the subjects in each weight group are shown in Table 2. Group differences (p < 0.05) were found for post-occlusive reactive hyperemic calf blood flow during the first 15 seconds following the occlusion test, as well as for the absolute and percentage changes in calf blood flow from rest to post-occlusive reactive hyperemia. All three measures were higher in the normal weight group than in the obese group (p < 0.017).

Table 2. Calf blood flow of normal weight, overweight, and obese subjects. The p value represents the significance among the three weight groups.

The calf blood flow measurements of the three groups adjusted for age, hypertension and calf skinfold thickness are shown in Table 3. Group differences (p < 0.05) remained for adjusted values of absolute and percentage changes in calf blood flow from rest to post-occlusive reactive hyperemia. The adjusted absolute change in calf blood flow was lower (p < 0.017) in the obese group than in the normal weight group, and the adjusted percentage change in the obese group was lower (p < 0.017) than in both the normal weight and overweight groups.

Table 3. Adjusted values of calf blood flow of normal weight, overweight, and obese subjects. The p value represents the significance among the three weight groups.

None of the anthropometric variables were significantly related (p > 0.05) to calf blood flow obtained at rest. BMI was inversely related to the post-occlusive reactive hyperemic calf blood flow (r = -0.25, p = 0.022), and to the absolute change (r = -0.44, p < 0.001) and percentage change (r = -0.37, p < 0.001) (Figure 1) in calf blood flow from rest to post-occlusive reactive hyperemia. Similarly, waist circumference was inversely related to post-occlusive reactive hyperemic calf blood flow (r = -0.21, p = 0.047), and to the absolute change (r = -0.36, p = 0.001) and percentage change (r = -0.32, p = 0.002) (Figure 2) in calf blood flow from rest to post-occlusive reactive hyperemia.

Figure 1. The relationship between body mass index and the percentage change in calf blood flow from rest to post-occlusive reactive hyperemia (r = -0.37, p < 0.001).

Figure 2. The relationship between waist circumference and percentage change in calf blood flow from rest to post-occlusive reactive hyperemia (r = -0.32, p = 0.002).

One subject had a percentage change in calf blood flow that exceeded 900%. We used two different approaches to assess whether this data point had an influence on the relationships shown in Figures 1 and 2. In the first approach, we used non-parametric procedures by calculating the Spearman Rank correlation coefficients. The percentage change in calf blood flow remained significantly related to BMI (r = -0.38, p < 0.001) and to waist circumference (r = -0.33, p = 0.002), which were similar to the coefficients obtained using parametric procedures. The second approach was to repeat the Pearson correlation coefficients after removal of this outlying data point. BMI remained inversely related to the post-occlusive reactive hyperemic calf blood flow (r = -0.23, p = 0.036), as well as the absolute change (r = -0.36, p = 0.001) and percentage change (r = -0.28, p = 0.014) in calf blood flow from rest to post-occlusive reactive hyperemia Additionally, waist circumference remained inversely related to the absolute change (r = -0.33, p = 0.002) and percentage change (r = -0.26, p = 0.019) in calf blood flow from rest to post-occlusive reactive hyperemia. The results from both approaches suggest that the outlying data point had minimal effect on the observed significant associations among these measures.

Discussion

This investigation compared the reactive hyperemic response to three minutes of arterial occlusion in older adults having a wide range in BMI, and determined if calf blood flow differences among normal weight, overweight, and obese older adults persisted after adjusting for confounders, such as age and hypertension. The primary findings were: (1) the obese group had a blunted change in post-occlusive reactive hyperemic blood flow, indicative of impaired vascular reactivity, than the normal weight group, and (2) the difference in vascular reactivity between the obese and normal weight groups remained significant after controlling for age, hypertension and calf skinfold thickness.

The observation that obesity impairs vascular reactivity supports previous studies in young and healthy adults [8,16,21], and extends this finding to obese older adults who are free of overt cardiovascular disease. This suggests that obesity-mediated vascular dysfunction in older adults is due to impairment in endothelial function [8,16]. Our results are further supported by a report that found an inverse correlation between obesity and endothelial-independent vasodilation [22] in a small sample of older adults with diabetes. Collectively, these findings suggest that obesity has a detrimental impact on endothelial function in older adults with and without diabetes.

Besides the negative consequences of obesity on vascular function in older populations, obesity-mediated alterations in endothelial function are evident even in young adults. A lower response in endothelial-dependent vasodilation and forearm blood flow after an infusion of acetylcholine (ACh) is observed in obese, young adults compared to overweight and normal weight young adults [8]. Accumulation of abdominal fat is the primary factor for endothelial dysfunction [21]. Interestingly, endothelial function may be positively altered following a weight loss program. A prospective investigation found ACh-stimulated forearm blood flow improved following a reduction in body size and waist circumference [16]. In speculation, a program designed to decrease adiposity may improve endothelial function in older, obese adults.

The increased prevalence of chronic vascular complications consistently shown in the aging population [23,24] further complicates the association between obesity and endothelial function. The development of endothelial dysfunction and, ultimately, atherosclerosis has specifically been linked to diabetes [25], hypertension [26], and hypercholesterolemia [27], all of which increase in prevalence with advancing age [28]. Additionally, lifestyle behaviors such as physical inactivity and smoking, impair endothelial function and initiate atherosclerotic processes [23,24,29-31]. The lack of control of these co-morbid conditions leaves previous results inconclusive regarding the independent role of obesity in the pathogenesis of endothelial dysfunction. The current investigation attempted to minimize these confounding factors by excluding subjects with cardiovascular disease, diabetes, and a history of smoking during the previous year. Furthermore, vascular reactivity measurements were adjusted for group differences in age, prevalence of hypertension and calf skinfold thickness. These approaches improve the ability to determine the influence of obesity on vascular reactivity.

Finally, subcutaneous body fat, as assessed by the sum of skinfolds, did not show any relationship to blood flow or to vascular reactivity in this population. In contrast, the central distribution of adipose tissue, as assessed by waist circumference, was inversely related to vascular reactivity of these older adults. Taken collectively, these results suggest that visceral adiposity may have a more detrimental influence on vascular reactivity (i.e., endothelial dysfunction) than subcutaneous fat. Our findings are supported by a previous observation that impairment in flow-mediated, endothelium-dependent vasodilation of the brachial artery occurs with visceral obesity, rather than with subcutaneous obesity [32].

One limitation to this study is the cross-sectional design which does not establish a causal link between obesity and impaired vascular reactivity. Intervention and longitudinal studies that track changes in fat mass and vascular reactivity are necessary to better determine their association. Although we normalized the blood flow measures according to subcutaneous fat of the calf, as estimated by the calf skinfold, we did not measure intra-muscular fat. Therefore, it is possible that the difference in blood flow among the groups is partially attributed to differences in intra-muscular fat of the calf. Additionally, the measurement of vascular reactivity by the method of reactive hyperemia assesses the vasodilatory function of both the endothelium and vascular smooth muscle, and therefore is only an indirect assessment of endothelial function. The lack of physical activity measurement is another limitation to this study. Physical activity status is associated with blood flow [24] and endothelial-dependent vasodilatory function [29] and, thus, should be considered in future studies examining the influence of obesity and abdominal fat on vascular reactivity.

Although our investigation minimized confounding factors by excluding for cardiovascular disease and controlling for many primary risk factors of atherosclerosis, we did not adjust for the possible influence of secondary risk factors, such as C-reactive protein or other inflammatory markers [33,34]. Furthermore, a medical history was used to exclude participants with diagnosed cardiovascular disease and diabetes, but those with undiagnosed conditions may not have been identified. That said, the possibility exists that our self-report method of revealing existing disease does allow for an underestimated prevalence of diabetes. Lastly, our assessment of adiposity was limited to BMI and anthropometric measures rather than more precise measurements of body fat and displacement of adiposity.

In conclusion, obesity and abdominal adiposity impair vascular reactivity in older men and women, and these deleterious effects on vascular reactivity are independent of conventional risk factors. Consequently, impaired vascular reactivity may increase the risk for subsequent cardiovascular complications in older, obese adults.

Competing interests

The author(s) declare that they have no competing interests.

Authors’ contributions

LSA conceived and designed the study, acquired data, performed the statistical analyses and their interpretation, and drafted each manuscript version. PCC, TLW and PSM assisted with subject recruitment and manuscript revision. KJN, ASF, CF assisted with data collection and manuscript revision. AWG conceived and designed the study, assisted with statistical analyses and their interpretation, and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This research was supported by grants from the National Institute on Aging (NIA) (R01-AG-16685; AWG), by a University of Oklahoma Research Council grant (AWG), and by the University of Oklahoma Health Sciences Center General Clinical Research Center grant (M01-RR-14467) sponsored by the National Center for Research Resources from the National Institutes of Health. The authors gratefully acknowledge Donald E. Parker, Ph.D. of the Department of Biostatistics and Epidemiology at the University of Oklahoma Health Sciences Center for his consultation and review of the statistical analyses performed in this manuscript.

Abstract Studies in experimental models suggest that endothelium-derived nitric oxide is reduced with aging, and this circumstance may be relevant to atherogenesis. The aim of this study was to determine whether increasing age resulted in altered endothelium-dependent vasodilation in the forearm resistance vessels of healthy humans. Forearm blood flow was measured in 119 healthy subjects, aged 19 to 69 years, by venous occlusion plethysmography. Brachial artery infusions of methacholine chloride (0.03 to 10.0 µg/min) were used to assess endothelium-dependent vasodilation and of sodium nitroprusside (0.03 to 10.0 µg/min) to assess endothelium-independent vasodilation. The slope of the dose–blood flow response relation was calculated in each subject for each drug. Univariate and multiple stepwise regression analyses were used to relate vascular reactivity to selected variables, including age, lipids, and blood pressure. Endothelium-dependent vasodilation was progressively impaired with increasing age, assessed as a reduction in slope from 2.25±0.16 to 0.34±0.11 (mL/100 mL tissue per minute)/(µg/min) (P<.001). The decline in endothelium-dependent vasodilation was already evident by the fourth decade (age 30 to 39 years). Endothelium-independent vasodilation did not change with age. Age, total cholesterol, and low-density lipoprotein cholesterol were univariate predictors of endothelium-dependent vasodilation. Age remained the most significant predictor of endothelium-dependent vasodilator responses by multiple stepwise regression analysis. From these observations, it can be concluded that endothelium-dependent vasodilation declines steadily with increasing age in healthy human subjects. Age is a strong univariate and multivariate predictor of endothelium-dependent vasodilation. This finding may be a marker for more widespread endothelial dysfunction.

Key Words: aging • vasodilation • nitric oxide • atherosclerosis

Introduction

Top

Abstract

Introduction

Methods

Results

Discussion

References

The prevalence of atherosclerosis increases in the elderly population, and the incidence of myocardial infarction, stroke, and limb ischemia is high in older individuals even after controlling for the presence of other cardiovascular risk factors, such as hypercholesterolemia, cigarette smoking, and hypertension.1 2 3 We have postulated that the aging blood vessel is less able to protect itself from injury, a situation analogous to that which occurs in patients with diseases such as hypercholesterolemia and diabetes. Specifically, we have hypothesized that the availability of ED-NO is reduced in older individuals. ED-NO is synthesized in endothelial cells from its precursor L-arginine by the enzyme NO synthase and induces vasorelaxation by activating guanylate cyclase on vascular smooth muscle. NO not only regulates vascular tone4 5 but also inhibits platelet aggregation, leukocyte adhesion to the endothelial surface, and vascular smooth muscle proliferation, properties that maintain vascular homeostasis and reduce injury.4 5 6 7 8 Therefore, limited availability of ED-NO may contribute to atherogenesis.

A number of studies in experimental models in animals suggest that the release or activity of ED-NO is reduced with aging.9 10 Furthermore, in humans, aging is associated with abnormal endothelium-dependent vasodilation to agents that stimulate the release of ED-NO, such as acetylcholine.11 12 13 Age appears to be a predictor of impaired endothelium-dependent vasodilation of epicardial coronary arteries, coronary resistance vessels, and peripheral conduit vessels.11 12 13 14 Nonetheless, although some of these observations are made in the presence of vascular disease or lipid disorders or with small sample sizes, this finding is not unlike those observed in disease states associated with atherosclerosis, such as hypercholesterolemia, diabetes, and hypertension.15 16 17

The purpose of this study was to determine whether endothelium-dependent vasodilation decreases in limb resistance vessels of humans who are otherwise healthy. We chose to study limb resistance vessels because these are not typically affected by atheroma, and abnormalities in these vessels would give evidence to the premise that impaired endothelial function is a diffuse process in the elderly. Furthermore, we enrolled a large number of subjects to ensure adequate statistical power for the study of age-related changes in vascular reactivity.

Methods

Top

Abstract

Introduction

Methods

Results

Discussion

References

Subjects

Studies were performed on 119 healthy subjects (62 men and 57 women) who ranged in age from 19 to 69 years. Each subject was evaluated by history, physical examination, and screening laboratory tests. No subject had evidence of hypercholesterolemia, diabetes, or hypertension; smoked cigarettes; or had any history of cardiovascular disease. No subject was taking diuretics, vasoactive medications, or nonsteroidal anti-inflammatory medications. These subjects served as a control population for a number of research protocols conducted in this laboratory, all of which had the approval of the Human Research Committee of Brigham and Women’s Hospital. No subject received any pharmacological intervention before administration of methacholine and nitroprusside. Written informed consent was obtained from each subject.

Experimental Protocol

Each subject was studied in a 22°C temperature-controlled room in the postabsorptive state. Alcohol and caffeine were prohibited within 12 hours of the study. The brachial artery of each subject was cannulated with a 1.5-in polyethylene catheter under sterile conditions with the use of local anesthesia. The indwelling arterial cannula was used for BP measurements and vasoactive drug infusions. The vascular research laboratory was kept quiet, and the lights were dimmed. All subjects rested for at least 30 minutes after catheter placement for establishment of a stable baseline before data collection.

For assessment of endothelium-dependent vasodilation, methacholine chloride was administered via the brachial artery in increasing doses ranging from 0.03 to 10.0 µg/min. For assessment of endothelium-independent vasodilation, intra-arterial infusion of sodium nitroprusside was administered at doses ranging from 0.03 to 10.0 µg/min. This agent acts directly on vascular smooth muscle by stimulating soluble guanylate cyclase and inducing hyperpolarization. Each drug was delivered at a rate of 0.4 mL/min. FBF measurements were made under baseline conditions until stability was assured and then during the 3rd to 5th minutes of infusion of each drug. Basal conditions were reestablished between drug infusions. The doses of each drug were chosen to achieve increases in FBF without causing systemic effects.

Bilateral FBF was determined by venous occlusion strain-gauge plethysmography using calibrated mercury-in-Silastic strain gauges and was expressed as milliliters per 100 mL of tissue per minute (DE Hokanson, Inc). Each arm was supported above heart level. Venous occlusion pressure averaged 35 mm Hg. Circulation to the hand was stopped by inflation of a wrist cuff to suprasystolic pressure before each FBF determination. Each FBF determination comprised at least five separate measurements performed at 10- to 15-second intervals. The direct effect of a vasoactive drug was determined by measurement of blood flow in the infused arm. One can ascertain that systemic effects have not occurred if blood flow in the contralateral forearm does not change during the infusion. Forearm vascular resistance was calculated as the ratio of mean BP to FBF and expressed as millimeters of mercury per milliliter per 100 mL tissue per minute. BP was measured via an indwelling arterial cannula attached to a Gould P23 pressure transducer aligned to an amplifier on a Gould physiological recorder.

The slope of the dose–blood flow response relation to drug infusion was calculated with a least-squares linear regression analysis for each drug infusion in each subject and expressed as (milliliters per 100 mL tissue per minute) per (micrograms per minute). A linear relationship was evident within the dose ranges used in these studies. An example of an individual slope calculation is shown in Fig 1. In this manner, the dose-response relationship for each drug in each subject was characterized by its slope.

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[in a new window] Figure 1. Sample dose-response curve relating FBF to increasing doses of a vasoactive drug. In this individual, the slope of the dose-response relationship is 0.78 (mL/100 mL tissue per minute)/(µg/min).

Statistical Analysis

Values are presented as mean±SE. Subjects were grouped by age into 10-year intervals, and ANOVA for repeated measures was used to compare the means of the groups. If a difference was detected among the means, Dunnett’s test was used to identify significant differences between means. The association between the continuous variables of age and the dose response was evaluated by linear regression analysis. In addition, univariate analysis of the effects of vascular risk factors and the methacholine response relationship for continuous variables (age, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, body mass index, mean BP, heart rate) was performed with linear regression. The interaction between these risk factors and the methacholine dose-response slope was then examined with multiple stepwise regression analysis run both forward and backward.18 Correlation coefficients were determined for age and the following variables: total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, weight, and mean arterial pressure. Statistical significance was accepted at a value of P.05.

Results

Top

Abstract

Introduction

Methods

Results

Discussion

References

Basal hemodynamic measurements including FBF, forearm vascular resistance, and mean BP for each decade of age are provided in Table 1. These variables did not differ significantly among the groups comprising each decade. The subjects aged 60 to 69 years appeared to have the lowest mean FBF and mean BP as well as the highest forearm vascular resistance, but these values did not differ significantly from those of the other decade intervals. Total, LDL, and HDL cholesterol concentrations, triglyceride concentrations, weight, and heart rate did not significantly differ among the age groups.

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[in a new window] Table 1. Baseline Hemodynamic Measurements by Decade

Endothelium-Dependent Vasodilation

Endothelium-dependent vasodilation was defined as the slope of the FBF response in milliliters per 100 mL tissue per minute to methacholine dose in micrograms per minute. This slope decreased significantly with each decade of age (P<.001 by ANOVA, Fig 2). Indeed, endothelium-dependent vasodilation decreased progressively with each decade studied (a significant difference was detected between all the means). Even by the fourth decade (30 to 39 years), the methacholine dose-response relationship decreased significantly from 2.25±0.16 (in the third decade) to 1.46±0.10 (mL/100 mL tissue per minute)/(µg/min) (P<.05). Subsequently, the slope decreased further to 1.05±0.18, 0.48±0.06, and 0.34±0.11 (mL/100 mL tissue per minute)/(µg/min) for the fifth, sixth, and seventh decades, respectively. A significant relationship between the methacholine response and exact age was also detected by linear regression analysis (Fig 3a), confirming that endothelium-dependent vasodilation declines progressively with aging (R=-.81, P<.001). The impairment in endothelium-dependent vasodilation with advancing age occurred irrespective of sex, being evident in both men and women.

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[in a new window] Figure 2. Slope of the methacholine dose response for each of the five decades studied. Endothelium-dependent vasodilation declined with each decade. Values represent mean±SE.

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[in a new window] Figure 3. Linear regression analyses depicting age vs slope of the dose-response relationship for methacholine (a) and nitroprusside (b) for each individual.

Endothelium-Independent Vasodilation

Endothelium-independent vasodilation was defined as the slope of the FBF response in milliliters per 100 mL tissue per minute to nitroprusside dose in micrograms per minute. In contrast to the findings with methacholine, the slope of this dose-response relationship did not change significantly with each decade of age (Fig 4). The mean slope was 1.11±0.21, 0.79±0.11, 0.76±0.13, 0.81±0.21, and 0.90±0.26 for the third, fourth, fifth, sixth, and seventh decades, respectively (P=NS by ANOVA). Linear regression demonstrated no significant relationship between endothelium-independent vasodilation and age (P=NS, Fig 3b).

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[in a new window] Figure 4. Slope of the nitroprusside dose response for each of the five decades studied. Endothelium-dependent vasodilation did not change significantly. Values represent mean±SE.

Univariate and Multivariate Models

To further evaluate the decline in endothelium-dependent vasodilation with advancing age, we evaluated relevant associations with the blood flow response to methacholine by univariate analysis (Table 2). Age was a strong univariate predictor of the FBF response to methacholine. Even though all subjects had total cholesterol less than 5.18 mmol/L and LDL cholesterol measurements less than the 75th percentile for their age and sex, both total cholesterol and LDL cholesterol were also univariate predictors of the endothelium-dependent response. There was no association, however, between endothelium-dependent vasodilation and mean BP or heart rate in these healthy subjects. We then included these variables in a multivariate stepwise regression analysis of methacholine response to determine whether age remains a significant predictor of methacholine response in their presence. In this stepwise multiple regression model and all the models we used (Table 3), age remained the most significant predictor of endothelium-dependent vasodilation.

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[in a new window] Table 2. Univariate Associations With Methacholine Response

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[in a new window] Table 3. Multivariate Analysis of Methacholine Response

Discussion

Top

Abstract

Introduction

Methods

Results

Discussion

References

The important finding in this study is that endothelium-dependent vasodilation in forearm resistance vessels declines progressively with increasing age. This abnormality is present in healthy adults who have no other cardiovascular risk factors, such as diabetes, hypertension, or hypercholesterolemia. We found that impairment of endothelium-dependent vasodilation was clearly evident by the fourth decade. In contrast, endothelium-independent vasodilation did not change significantly with aging. These observations enable us to conclude that reduced availability of ED-NO may occur as humans age and speculate that this abnormality may create a vascular milieu that is conducive to atherogenesis.

Experimental Models of Aging

Our findings are supported by studies of vascular reactivity in experimental models of aging. In aged rats, vasodilator responses of cerebral arterioles to agonists that release endothelium-derived relaxing factor are reduced.9 In large cerebral arteries, the vasoconstrictor response to serotonin is increased significantly in aged rats when compared with younger adult rats.19 This may occur because the endothelium-dependent vasodilation to serotonin is diminished, allowing less opposition to its direct vasoconstricting action. Endothelium-dependent vasodilation also decreases with age in the rat aorta, femoral artery, and carotid artery.20 21 22 Also, endothelium-dependent vasodilation to acetylcholine but not endothelium-independent relaxation to an NO donor, decreases with increasing age in the resistance arteries of rats.23 Endothelial thickness is known to decrease with age in rats,24 which may partly explain these findings. In contrast, aging does not impair endothelium-dependent vasodilation in beagle hindlimb resistance vessels.25

Aging and Endothelial Function in Humans

Aging is a determinant of abnormal endothelium-dependent vasodilation in epicardial coronary arteries as well as in coronary resistance vessels of patients with multiple risk factors for atherosclerosis. Vita and colleagues11 demonstrated that increasing age was one predictor of abnormal endothelium-dependent vasodilation in atherosclerotic human epicardial coronary arteries. Similarly, Yasue and colleagues26 demonstrated impaired endothelium-dependent vasodilation of angiographically normal coronary arteries in subjects more than 30 years old, but 44% of these subjects had risk factors for atherosclerosis. Zeiher et al12 reported that age as well as total serum cholesterol levels were independent predictors of reduced endothelium-dependent vasorelaxation to acetylcholine in human coronary resistance vessels in vivo, but many of these patients had multiple risk factors for atherosclerosis. Similarly, aging-associated impairment in endothelium-dependent vasodilation in coronary resistance vessels has been described by Egashira et al13 in a small study of 18 subjects. Celermajer et al14 reported that aging is associated with impaired flow-mediated, endothelium-dependent vasodilation in the brachial artery. Progressive endothelial dysfunction in this conduit artery occurred even in the absence of cardiovascular risk factors. One small study did not find that age affects endothelium-dependent vasodilation of forearm resistance vessels, but it may have lacked sufficient statistical power.27 Our findings are similar to those recently reported by Taddei and colleagues,28 who evaluated endothelium-dependent responses to acetylcholine in both normotensive and hypertensive subjects.

In our study, increasing age was accompanied by a progressive decline in endothelium-dependent vasodilation in human limb resistance vessels, extending observations made in previous studies to resistance vessels of healthy subjects. Our findings are not confounded by the presence of atheroma or other risk factors for atherosclerosis, such as diabetes, hypercholesterolemia, and hypertension. This observation underscores the likelihood that diffuse and progressive impairment of endothelial function occurs with aging, creating a milieu that predisposes to vascular injury.

Potential Mechanisms of Aging-Induced Endothelial Dysfunction

The mechanism or mechanisms of endothelial dysfunction that occur with age have not been elucidated. Potential mechanisms include reduced synthesis and release of ED-NO, increased activity of vasoconstrictive prostanoids, poor diffusion of NO to smooth muscle because of increased intimal thickness, degradation of ED-NO by oxygen-derived free radicals, or advanced glycosylation end products.

Of these possibilities, we believe that the most plausible involve increased degradation of NO. Free radicals such as superoxide anion and hydrogen peroxide may decrease the half-life of released NO.29 Acute or cumulative response to environmental pollutants can expose the vessel wall to higher levels of free radicals.30 31 Oxidant stresses, and therefore free radical concentration, also may be greater in older individuals because of changes in diet.32 33 Advanced glycosylation end products increase with age and may contribute to decreased endothelium-dependent vasodilation by inactivating ED-NO, as they do with diabetes.34 35

Structural changes occur in the vasculature with aging and could contribute to altered vascular responses. These changes are particularly evident in large and medium-sized arteries36 and include decreased distensibility with age.37 Morphological changes occur in the media, where the orderly arrangement of laminae and elastin fibers is lost38 and elastin is replaced with collagen.39 These structural changes do not contribute to the findings in this study, as endothelium-independent vasodilation did not decline with aging.

Conclusion

Endothelium-dependent vasodilation becomes progressively impaired as individuals age. This abnormality occurs in resistance vessels and as such may be a marker for more widespread endothelial dysfunction. Therapeutic strategies directed at improving endothelial function should be studied because they may reduce the incidence of atherosclerosis that occurs with aging.

Selected Abbreviations and Acronyms

BP = blood pressure

ED-NO = endothelium-derived nitric oxide

FBF = forearm blood flow

HDL = high-density lipoprotein

LDL = low-density lipoprotein

NO = nitric oxide

Arasys Perfector, LLC is the Global distributor of the UK-based Anti-Aging Technology of Ion Magnum, Arasys, and Perfector. These three technologies share one vision: Working with the body’s own resources to prevent aging, enhance biological functions, well-being and longevity. Arasys Perfector, LLC has been conducting its own research and development since 2000. Most of our profit goes into an on-going effort of collaboration with anti-aging experts and doctors to improve the amazing capabilities of our micro/nano/pico current devices. Arasys Perfector, LLC began providing products, training, and solutions to businesses in the USA since 2004 and has begun partnership distributions in other parts of the world such as Asia, Canada, and Dubai starting in 2008.

We’ve developed this weblog to keep our current and prospective customers involved with the on-going research surrounding the Arasys, Perfector, and Ion Magnum devices.

Obesity is associated with more than 30 medical conditions including Type 2 Diabetes, Coronary Heart Disease, Osteroarthritis, High Blood Pressure, Breast Cancer, Cancers of the Esophagus and Gastric Cardia, Impaired Immune Response. Low Back Pain etc. Selim et al (2008) have shown that Obesity is related to reduced blood flow velocities in the middle cerebral arteries. Laasko et al (1990) has shown that reduced insulin-mediated glucose uptake in human obesity is due to defects in insulin’s action to increase blood flow to these tissues. Laasko et al report that this defect in insulin’s action is a novel mechanism of insulin resistance. Overall, obesity is characterized by decreased blood flow into muscle. The reduced blood flow and/or tissue activity can lead to decreased insulin-medicated glucose uptake, another factor associated with obesity according to Laasko et al (1990). Cheuk-Kwan Sun et al (2003) demosnstrated that obesity is related with reduced portal venus blood flow, and a decrease in overall hepatic perfusion and oxygenation.

A clinical study with individuals presenting abnormally clumped Red Blood Cells’ (RBCs) was completed in February 2009 with a device representing the Pacemaker Technology for the Skeletal muscle (PTSM / Ion Magnum - IM). Results (see figure 1) indicate that this technology rapidly and efficiently leads to normalized erythrocytes’ separation at the microscopic level. RBCs separation is crucial for the overall blood flow and timely transport of hormones, antibodies, oxygen and nutrients to the cells, and waste products to the kidneys. Transport of Hormones is a crucial process lipolysis (T3 and Growth Hormone — GF) and muscle hypertrophy (Insulin Growth Factor - IGF-1). Additionally, erythrocyte separation resulting from treatment with the Pacemaker technology for the skeletal muscle appears to have a negative correlation with the number of fungal forms, poikilocytosis, thrombocyte aggregation and bacteria present in the blood prior to the IM treatments, In summary, the erythrocyte separation resulting from treatments with the pacemaker technology for the skeletal muscle enhance hormonal transport including T3 and GH enhances hormonal transport including T3 and GH leading to lipolysis and muscle hypertrophy; 2) RBC;s separation enhances overall level of health by a significant reduction of free radicals. bacteria, fungal forms. etc.; 3) Obesity is characterized by reduced blood flow. PTSM increases RBC’s separation resulting in normalized blood flow. In conclusion, re-establishing normal levels of blood flow will not only help reduce obesity but it will help reduce the risk of heart attack as well as all other disorders associated with obesity.

The fact that the Pacemaker Technology for the Skeletal muscle reduces Obesity is shown in a recent clinical study 2009. Five separate clinics from around the world participated in this clinical study . All five subjects that participated in the study showed substantial weight loss, including reduction of visceral fat. Jensen (2008) reports that an upper body/visceral fat distribution in obesity is closely linked with metabolic complications, whereas increased lower body fat is independently predictive of reduced cardiovascular risk. The before and after of subject 3 are shown in figure 2.

IM Research, the Pacemaker Technology, London University, UK Xanya Sofra-Weiss, Ph.D

The Pacemaker Technology for the Skeletal Muscle (PTSM) is a voltage driven device, very much like the Pacemaker. However, due to the complexity of the CNS, PTSM is based on a dynamic multi-sine, analogue waveform that was originally tested at the cellular level by Dr. Donald Gilbert, a molecular biologist, in the eighties. After 30 years of research, the IM was electronically engineered by the Co-Inventor of the first Pacemaker (2008) to resonate the motor nerve’s signal of strenuous exercise normally emitted by the brain. Due to its resonance with the biological signal, the PTSM signal spreads throughout the CNS inducing effortless and painless isometric and isotonic muscle contractions. The signal to the nerve ultimately triggers hormonal secretion such as Growth Hormone (GF), Thyroxine (T4) and Triiodothyronine (T3) for lipolysis and Insulin Growth Factor (IGF-1) for muscle hypertrophy.