The Role of Nitric Oxide in Cellular Aging:Telomeres, Mitochondria and Stem Cells

Nathan S. Bryan, Ph.D.Texas Therapeutics Institute

ACIM October 24-25, 2013

Structure of Presentation

What is Regenerative Medicine

Current Aging/Disease Hypotheses

Brief Overview of Nitric Oxide (NO)

NO effects on telomerase, mitochondria and stem cells

Strategies to diagnose and replete NO

Disclosure: N.S. Bryan is the Founder and Chief Science Officer of NeoGenis Labs, Inc.

Good Medicine – Applied Physiology

Bad Medicine – Applied Pharmacology

Chronic Diseasesaccount for 61% of deaths worldwide.

Most of these preventableby diet and lifestylemodifications.

Regenerative Medicine

process of replacing, engineering or regenerating human cells, tissues or organs to restore or establish normal function. This field holds the promise of engineering damaged tissues and organs via stimulating the body's own repair mechanisms to functionally heal previously irreparable tissues or organs.

What is Aging?

Aging is the biological consequence of our body’s inability to make new cells that work properly.

Jerry Tennant MD

1. What is needed to make new cells?

2. What makes them work properly?

What is needed to make new cells?

1. Fats and cholesterol for cell membrane20% of your body is fat (need the right fats)

2. Amino acidsneeded to make the protein machinery inside the cells

3. Vitamins and mineralsallow the body to make the fats and proteins work

What causes aging and is involvedIn regenerative medicine?

Three main hypotheses:

1. Telomere shortening2. Mitochondrial dysfunction3. Loss of stem cell function and repair

Unified Theory of Aging

Nitric Oxide controls and regulates1. Telomerase activity2. Mitochondrial biogenesis and function3. Mobilization of resident stem cells

Telomere Theory of Aging

A decrease in telomerase activity precedes telomere shortening andintroduction of telomerase into normal human cells extends life-span .Bodnar et al. Science 1998 Jan 16;279(5349):349-52.

On the cellular level, senescence, chromosome stability, and cell viability are regulated by the telomeres and their associated proteins, deoxyribonucleic acid-protein complexes located at both ends of eukaryotic chromosomes Blasco Nature Reviews Genetics 6, 611-622 (August 2005)

Shortening of the telomeres has been shown to be associated with increased mortality rate from age related diseases. Individuals with shorter telomeres had a mortality rate nearly twice that of those with longer telomeresCawthon et al Lancet 2003; 361: 393–95

Nitric Oxide Activates Telomerase and Delays Endothelial Cell Senescence

NO interferes with telomerase activity thereby inhibiting telomere shortening. The mechanism by which NO stimulates telomerase activity remains to be determined. Vasa et al Circulation Research. 2000; 87: 540-542

eNOS activity is required for hTERT expression and is dependent uponNO production. eNOS knockout mice lose regulation of telomerase activitythat is rescued by exogenous NO donorsGrasselli et al Circulation Research. 2008 Jul 3;103(1):34-42

NO is the master signaling molecule in the regulation of telomerase activityand provides the opportunity for innovative therapeutic approaches based onthe use of NO active compoundsFarsetti et al J. Appl Physiol. 2009 Jan;106(1):333-7

Schematic illustration of the mechanism involved in estrogen receptor- and endothelial nitric oxide synthase (eNOS)-induced hTERT transcription.

Farsetti A et al. J Appl Physiol 2009;106:333-337

Mitochondrial Theory of Aging

1. Free radicals play a major role in aging and most are mitochondrially produced.

2. Mitochondrial DNA lacks the protective DNA-binding protein (called histones), has less efficient DNA repair, and is close to the free radical-producing ETC, resulting in high levels of mitochondrial DNA damage compared to nuclear DNA.

3. Caloric restriction, the only known treatment to increase the mammalian lifespan, reduces mitochondrial free radical production and mitochondrial DNA oxidative damage.

4. Fibroblasts injected with mitochondria from old rats degenerate more rapidly than fibroblasts injected with mitochondria from young animals.

5. The mitochondria of older mammals are often larger and less efficient than those from younger mammals.

6. Targeted increased mitochondrial catalase (an enzymatic anti-oxidant) expression increases the mouse lifespan by around 20%.

7. Studies between different species have demonstrated that longer-lived species typically have lower mitochondrial DNA oxidative damage and lower free radical production. 

8. Targeted mutation of the mitochondrial DNA polymerase-g, which causes an increased mitochondrial mutation rate with aging, results in a premature aging phenotype.

Mitochondria – Cellular Power Plants

Mitochondria are found in nearly all eukaryotes. They vary in number and location according to cell type. A single mitochondrion is often found in unicellular organisms. Conversely, numerous mitochondria are found in human liver cells, with about 1000–2000 mitochondria per cell, making up 1/5 of the cell volume.

Generate ATP, used as a source of chemical energy

Involved in cell signalingHeat ProductionCellular metabolismCellular differentiationCell deathControl of the cell cycle (cancer)Cell growthSteroid synthesis

Mitochondria have been implicated in many human diseases and arecritical in the aging process.

Nitric Oxide Controls and RegulatesMitochondrial:

ATP synthesisReactive Oxygen SpeciesCell SignalingApoptosis (Cell Cycle)BiogenesisMetabolism/Bioenergetics

Nitric Oxide and Mitochondrial Biogenesis

Nisoli E et al. Circulation Research 2007;100:795-806

Copyright © American Heart Association

Nitrite-dependent extension of oxygen gradients. (A) During normoxia, NOS is functional, myoglobin is oxygenated, and sufficient oxygen is available to diffuse from the source of oxygen through the tissue. (B) In hypoxic conditions, NOS is substrate limited and cannot make NO and myoglobin becomes deoxygenated. The majority of oxygen present is consumed by mitochondria close to the oxygen source, leading to a shortened oxygen gradient. (C) If nitrite is present during hypoxic conditions, it can be reduced by deoxygenated myoglobin. The NO generated can then partially inhibit mitochondrial oxygen consumption, allowing more oxygen to diffuse past these mitochondria and further into the tissue (elongation of oxygen gradient).

Extension of Tissue Oxygen Gradients and Modulation of Exercise Capacity by

Nitrite Reduction to NO

Shiva S. Nitric Oxide 2010 Feb 15;22(2):64-74

Nitrite regulates mitochondrial function. During hypoxia, nitrite is reduced to NO by deoxygenated myoglobin and nitrosylates the binuclear center of complex IV. This results in the inhibition of oxygen consumption which may contribute to the regulation of oxygen gradients and the modulation of exercise efficiency. During ischemia/reperfusion, nitrite is converted to a nitrosating species (possibly N2O3 through its reductive anhydrase reaction with heme) and S-nitrosates complex I at reperfusion. This leads to decreased ROS generation and inhibition of cytochrome c release, which contribute to cytoprotection after I/R.

Nitrite Regulates Mitochondrial FunctionAt Different Stages of ETC

Stem Cell Theory of Aging

Aging is the result of the inability of various types of stem cells to continue to replenish the tissues of an organism with functional differentiated cells capable of maintaining that tissue’s original function.

The number of stem cells in young people is very much higher than older people and this cause a better and more efficient replacement mechanism in the young contrary to the old.

Aging is not a matter of the increase of damage, but a matter of failure to replace it due to decreased number of stem cells. They decrease in number and tend to loose the ability to differentiate

Nitric Oxide is the requisite signal for stem cell mobilization and differentiation

into target cell types

The bioavailability of NO in patients may predict stem cell therapy success or failure

Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cellsAicher et al Nature Medicine 9, 1370 - 1376 (2003)

Nitric oxide-cyclic GMP signaling in stem cell differentiation.Free Radic Biol Med. 2011 Dec 15;51(12):2150-7

Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells.

Mujoo K, Sharin VG, Bryan NS, Krumenacker JS, Sloan C, Parveen S, Nikonoff LE, Kots AY, Murad F.

Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18924-9

What is Nitric Oxide?

The chemical compound nitric oxide is a gas with chemical formula NO٠.

It is an important signaling molecule in the body of mammals including humans, one of the few gaseous signaling molecules known.

It is also a toxic air pollutant produced by automobile engines and power plants.

NO should not be confused with nitrous oxide (N2O), a general anesthetic, or with nitrogen dioxide(NO2) which is another poisonous air pollutant.

The nitric oxide molecule is a free radical, which is relevant to understanding its high reactivity. It reacts with the oxygen in air to form nitrogen dioxide, signaled by the appearance of the reddish-brown color.

NO

Cardiovascular System

Respiratory Tract

Immunology

Cell Proliferation

Central Nervous System

Peripheral Nervous System

Gastrointestinal/Urogenital Tract

VasorelaxationBlood Cell RegulationMyocardial ContractilityMicrovascular Permeability

BronchodilatationAsthma, ARDS

NANC nerve-mediatedRelaxation

Learning and MemoryPain SensitizationEpilepsyNeurodegenerationCentral BP Control

ApoptosisAngiogenesisTumor Cell Growth

Unspecific ImmunityInhibition of Viral ReplicationTransplant Rejection

Penile ErectionPre-term Labour

Nitric Oxide Plays a Key Role in the Regulation of Numerous Vital Biological Functions

Regeneration

Mobilization of resident stem cellsTargeted differentiation

NOS

L-citrulline

L-arginine

NOENDOTHELIUM

Shear Stress ACH

M

SMOOTHMUSCLE

Relaxation

GTPcGMP

NO

guanylylcyclase (inactive) guanylyl

cyclase (active)

+PD5 inhibitors

L-Arg

Diet

L-Arg

ArginaseADMA

Transport

NOSUncouplingReduced OxygenReduced Cofactor + SubstrateOxidative Stress

Antioxidants

NO2

NO3

Oxidation

Bacterial Reduction

NO

+

O2-٠

ONOO-

Health

Disease

BH4 Ca/CamFAD+ FMNNADPH O2Heme iron GSH

MitochondriaXONADPH oxidase

The L-Arginine-Nitric Oxide Pathway

Urea Cycle

L-Arg

10 20 30 40 50 60 70

0

20

40

60

80

100

% D

eclin

e in

NO

Pro

duct

ion

Age in years

men women

Gerhard et al Hypertension 1996Celermajer et al JACC 1994Taddei et al Hypertension 2001Egashira et al Circulation 1993

Humans lose abilityto produce NO with aging

What if 50% or more of NObioactivity was determined and

dictated by foods and diets containingnitrite and nitrate?

Atmospheric Nitrogen CycleThe store of nitrogen found in the atmosphere, where it exists as a gas (mainly N2), plays an important role for life. Most plants can only take up nitrogen in two solid forms: ammonium ion (NH4+ ) and the nitrate ion (NO3- ). Most plants obtain the nitrogen they need as nitrate from the soil. When released, most of the ammonium is often chemically altered by a specific type of bacteria (genus Nitrosomonas) into nitrite (NO2- ). Further modification by another type of bacteria (genus Nitrobacter) converts the nitrite to nitrate. All nitrogen obtained by animals can be traced back to the eating of plants at some stage of the food chain.

Dietary nitrate is rapidly absorbed into the bloodstream, where it mixeswith endogenous nitrate from the NOS/NO pathway. A large portion of nitrate is taken up by the salivary glands, secreted with saliva and reduced to nitrite by symbiotic bacteria in the oral cavity. Salivary-derived nitrite is further reduced to NO and otherbiologically active nitrogen oxides in the acidic stomach. Remaining nitrite is rapidly absorbed and accumulates in tissues, where it serves to regulate cellular functions via reduction to NO or possibly by direct reactions with protein and lipids. NO and nitrite are ultimately oxidized to nitrate, which again enters the enterosalivary circulation or is excreted in urine.

New Paradigm - Human Nitrogen Cycle

One-electron reduction is favorable to five-electron oxidation

Nitrate, bacteria and human healthLundberg JO, Weitzberg E, Cole JA, Benjamin N.Nat Rev Microbiol. 2004 Jul;2(7):593-602

Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite.Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia A.Hypertension. 2008 Mar;51(3):784-90.

Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans.Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, Benjamin N, Jones AM.J Appl Physiol. 2009 Oct;107(4):1144-55.

Physiological role for nitrate-reducing oral bacteria in blood pressure controlKapil V, Haydar SM, Pearl V, Lundberg JO, Weitzberg E, Ahluwalia A.Free Radic Biol Med. 2013 Feb;55:93-100.

Dietary Nitrate Can Be Metabolized to Nitrite and NO

NO 3-

NO 2-

NO

N 2O

N 2

NH 3

2e-

1e-

1e-

1e-

3e-

Bacteria

Increase NO 2-

NR

NiR

NOR

N 2OR

Ideal Community:• Higher Nitrate reduction efficacy• No NiR enzyme; Nitrite can accumulate, enrich saliva to form NO when swallowed.

Best

Intermediate

Worst

Hyde et al PLoS One (in press)

NEW PARADIGM FOR NO REGULATION

- There exists specific bacterial communities that provide the human body with continuous sources of nitrite and NO from dietary nitrate.

- Contributes to optimal cardiovascular health

- Absence of these oral bacterial communities affects NO homeostasis.

- Individuals deficient in these commensal bacteria would be NO deficient and perhaps at increased risk for cardiovascular disease

Disruption of Nitrate-Nitrite-NO Pathway

1. Insufficient dietary intake of nitrate/nitrite rich foods(green leafy vegetables, beets, etc)

2. Problems with nitrate uptake in duodenum (sialin (SLC17A5) transporter mutations – Salla Disease)

3. Insufficient saliva production (Sjogrens syndrome)

4. Lack of oral commensal bacteria to reduce nitrate to nitrite (use of antibiotics/antiseptic mouthwash, poor oral hygeine)

5. Insufficient stomach acid production – Achlorhydria(use of PPI’s, H. Pylori infection, iron overload)

6. Increased oxidative stress that scavenges NO

What might this mean?

• Absence of these select bacteria - a new risk factor for cardiovascular disease.

• Patients with periodontal disease , affecting the NO producing communities - possibly linking oral health to cardiovascular disease risk by disruption of NO production

• Use of antiseptic mouthwash or overuse antibiotics can disrupt nitrate reducing communities

• Patients taking proton pump inhibitors to suppress stomach acid production

• Develop this pathway as a primary therapeutic target to affect NO production

NO3-

NO2-

NO

Manipulating the NO System Through Diet and Nutrition

oxidation reduction

Facultative anaerobes5-8%Spiegelhalder 1976Lundberg 2004

Mammalian enzymes~ 0.01%Bryan Nat Chem Biol. 2005Feelisch JBC 2008

Beet, kale, etc

Oxygen,ceruloplasmin

Oxyhemeproteins

L-arginine

50-90%

Lowering blood pressure by 5 mmHgreduces risk of stroke by 34% and

Ischemic heart disease by 21%

Health Technol Assess. 2003;7(31):1-94.

Lowering blood pressure to prevent myocardial infarction and stroke: a new preventive strategy.

Law M, Wald N, Morris J.

What are the available strategiesFor enhancing NO?

Strong & sustained Nitric Oxide activity

NO based Clinical Trial Results

Zand et al Nutrition Research 2011

0 60 120 180 240 300 360 420 480 540 600

0

2000

4000

6000

8000

10000

12000

14000

NO

[ppb

]

Time (seconds)

0 10 20 30 40 50 60

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Blo

od N

O L

evel

s [µ

M]

Time (min)

L-Arginine + antioxidants Neo40 Daily

Pla

sma

nitr

ite [µ

M]

Day 0 Day 30

50

100

150

200

250

300

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400

Tri

gly

ceri

de

s (m

g/d

L)

A

Day 0 Day 30

0

50

100

150

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300

Tri

gly

ceri

de

s (m

g/d

L)

*p = 0.02

B

Significant reduction in patients with elevated triglycerides

Zand et al Nutrition Research 2011

Clinical Trial Results

Blood pressureUltrasoundPulse waveEndopat

0 10 min 20 min 30 min 60 min 4 hours

Ultra-sound BP

Pulsewave BP Endopat

Active

Placebo

Blood pressureUltrasoundPulse waveEndopat

0 10 min 20 min 30 min 60 min 4 hours

Ultra-sound BP

Pulsewave BP Endopat

3 week washout

Hypertension Study Protocol

Baseline 4 hour Neo0.0

0.5

1.0

1.5

2.0

2.5

3.0

End

osco

re

Endopat *d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

Hypertension Trial – Vanderbilt Univ

0 10 20 30 40 50 6080

85

90

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140

145

150

*

*

Blo

od P

ress

ure

(mm

Hg)

Time (min)

Systolic Active Diastolic Active Systolic Placebo Diastolic Placebo

#* @

#* @

@

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

d e m o d e m o d e m o d e m o

Houston, Hays JCH 2014

4 hour post active

0 10 20 30 40 50 60

80

85

90

135

140

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150

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160

*

*

*

*

Blo

od P

ress

ure

(mm

Hg)

Time (min)

Systolic Diastolic

Hypertensives n=17

0 10 20 30 40 50 60

80

85

90

125

130

135

140 Pre-Hypertensives n=9

*

*

*

*

Blo

od P

ress

ure

(mm

Hg)

Time (min)

Systolic Diastolic

0 10 20 30 40 50 60

80

85

90

115

120

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130

135Normotensives n=5

Blo

od P

ress

ure

(mm

Hg)

Time (min)

Systolic Diastolic

0 10 20 30 40 50 60

80

85

90

135

140

145

150

#*

*

*

Blo

od P

ress

ure

(mm

Hg)

Time (min)

Systolic Diastolic

* #

n=30

Representative Ultrasound Before and10 minutes after NO

13% increase in vessel diameter causesa 34% increase in blood flow

0 2 4 6 8 10

0.50

0.55

0.60

0.65

0.70

0.75

0.80

LCCA d

iam

eter

(cm

)

Time (min)

N0 Dilates Carotid Artery within 90 Seconds

Houston, Hays JCH 2014

Average Changes in 10 subjectsAfter 10 minutes

Thermographic Images

Before NO 10 min After NO

49 yof chronic smoker with Raynauds

Pre-hypertension trialCedars Sinai Medical CenterPI: Ernst Schwarz MD, PhD

  Group 1 (n=17) Group 2 (n=12)

Males 10 5

Females 7 7

Age 39 ± 11.77 43 ± 10.68

Blood Pressure (mmHg) 138 ± 11.66 (systole), 84 ± 5.09

(diastole)

138 ± 21.37 (systole), 80 ± 7.68

(diastole)

Heart Rate (bpm) 74.7 ± 9.24 80.4 ± 10.44

Orthostatics (mmHg) 139 ± 10.95 (systole), 86 ± 4.12

(diastole)

134 ± 19.22 (systole), 82 ± 7.79

(diastole)

Baseline Demographics

Pre-Hypertension Trial – Cedars SinaiSchool of Medicine

160 160

135 135

90 90

45 45

Group 1Baseline Follow Up

Group 2Baseline Follow Up

mm

Hg

mm

Hg

Blood PressureFigure 1

Systole

Diastole Diastole

Systole

Biswas et al (in press) JCPT

  Group 1 (mean ± SD) Group 2 (mean ± SD) Baseline: NO vs

placebo (p-value)

Follow-Up: NO vs placebo (p-value)

Baseline Follow-Up ∆ Baseline Follow-Up ∆

BP (mmHg, systole; diastole)

138±12; 84±5

126±12; 78±4 12; 6 reduction (p<0.001)

138±21; 80±8

135±17; 82±8

N.S. 0.19; 0.012

0.26; 0.25

Heart Rate

(bpm)

75±9 76±8 N.S. 80±10 79±8 N.S. 0.14 0.33

6-Minute Walk Test (meters)

596±214 650±197 55 improvement (p<0.005)

590±8 606±225 N.S. 0.25 0.35

SF-36v2 (PCS; MCS)

48±10; 40±9

50±8; 45±7 p<0.05 43±10; 37±9

37±11; 37±7

significant worsening (p<0.05)

0.08; 0.06 0.08; 0.03

30 Day Placebo controlled Trial

Biswas et al (in press) JCPT

Baseline 6 months

500

550

600

650

700

750

800

850

CIM

T (m

icro

ns)

Edwin Lee MD – case report

NO Leads to Plaque Regression

NO Supplementation Rescues Inborn Error in Metabolism

The Urea Cycle converts ammonia to urea for excretion

• Hyperammonemia • In addition:

– Progressive liver dysfunction and cirrhosis– Coagulopathy– Neurological dysfunction independent of

recurrent hyperammonemia– Hypertension– Renal dysfunction

• More than hyperammonemia?

ASL deficiency is an Inborn error in metabolism

3/22—116/753/23—122/783/24—133/803/25—106/803/28—120/753/29—114/773/30—124/723/31—126/73 4/1—109/80

NO

NO

NO

CONCLUSIONS

Nitric oxide controls and regulates telomeres, mitochondria and stem cell function

There is an age-related decline in NO production that asserts its effecton all 3 theories of aging

Restoring NO production can lead to better mitochondrial function, increasedtelomerase activity and improved mobilization of stem cells for tissue repair

Strategies to restore NO production/homeostasis will have a profoundimpact on public health and on the aging process

Any anti-aging strategy should include NO as a first line of defense.

Beware of Pretenders!!!

1. Ask for clinical evidence that NO productswork

2. Make them show you it works

3. Demand published research on the product

If they cannot provide you these 3simple requests, then RUN

Nitrosating Agents

Mex+NO

NO2

L-Arg

O2

L-NIO

Nitrosation

Nitr

osy

latio

n

Cellular Targets

Nitroso/NitrosylProducts

For

mat

ion

of

Nitr

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Nitr

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Spe

cies

NO

-Gen

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ion/

Det

ectio

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ox.red.

Transnitrosation

For

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itric

Oxi

deO2• -

ONOO -

NO3-

+H+

- OH•

NO2-

H2Oox

.

ox.

[NO+] Amines Metals

RSNO R2NNO

NO

Hypoxia / H+

Thiols

red.

N2O3

H2O

1:1

RSH R2NH Mex+

RSH

N2O4

RS•Oxi

da

tive

Nitr

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latio

n

-NO2-

Biochemical Pathways of NO-Target Interactions in vivo

Bryan et al., PNAS 2004

Book Highlights:Restoring nitric oxide

production in the body thereby combating:

• High blood pressure• Heart attack

• Stroke• Diabetes• Arthritis

• Kidney disease• Memory loss• Osteoporosis