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Dietary Protein and Bone HealthEmphasis: Animal Protein

Z.K. (Fariba) Roughead, PhD, RDResearch Nutritionist

USDA-ARSGrand Forks Human Nutrition Research Center

USDA-ARS Human Nutrition Research Centers

15 Senior Scientists135 support staff2 metabolic kitchensService labs: clinical lab, mineral analysis

2 DXAs, Whole body counter

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Osteoporosis affects 200 million worldwide

20% of elderly with hip fx die within the first year

Risk Factors for Osteoporosis

• Age• Gender• Genetics• Sedentary life style• Smoking• Alcohol• Low calcium / Vitamin D • High protein intake (NIH Consensus Statement, 2000)

The Controversy

High protein diets are a risk for osteoporosis…

Especiallyif source is animal protein

andcalcium intake is low

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The Evidence• Purified sources of protein

increased urinary calcium loss (Linkswiler et al 1970s)

• common sources of proteindid not (Spencer et al,1978; Hunt, et al 1995)

Phosphorus Effect?

Observational EvidenceFeskanich et al (1996): Animal protein associated

with increased risk of forearm fracturesMunger et al (1999): Animal protein associated

with reduced incidence of hip fracturesHannan et al (2000): Lower total and animal protein

intake associated with increased bone lossWengreen et al (2004): Increased total protein

intake associated with reduced hip fx in “younger” elderly

Bone• Composition:

– 70% mineral (40% calcium; 99% body Ca)

– 25% organic (>90% protein)

– 5-8% water• Function

– Structure, locomotion, hematopoesis, growth factors

– Acid-base balance

Renal Mechanisms for Acid-Base Balance

Animal Protein Intake

Sulfur Amino Acid Intake

Osteoclastic activity Bone Resorption

Renal Net Acid Excretion

Glomerular Filtration Rate

Bone Mineral Density

Renal Reabsorption of Calcium

Bone Resorption

Bone Mineral Density

Net Endogenous Acid

Research Questions …

Does increased animal protein intake decrease calcium retention? Does replacing animal protein with plant protein improve calcium retention? Do dietary calcium and protein interact in a dose-dependent manner?

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Design Considerations

• Purified vs. Common protein• Equalized P vs. Variable P• Urinary Ca vs. Ca retention• Balance vs. Tracer methodology

(absorption vs. retention)

• Short-term vs. Long-term feeding• Few subjects vs. Statistical power

Protocol• Volunteers: - healthy post-menopausal

women • Design: - randomized crossover• Diets: - weighed diets; 7-8 wk • Calcium Retention:

- wk 3-4: labeled entire 2-d menu with 47Ca (constant specific activity)

- whole body scintillation counting for 4 wk

Diet Composition1

Low Meat High Meat

Meat (beef, chicken, turkey, pork), g 65 112Protein, % of energy 12 20

g/kg bw 0.94 1.62

Fat, % of energy 30 31Carbohydrate, % of energy 58 49Calcium, mg 700 (594.2 ± 19.3) 698 (574.1 ± 18.4)

Phosphorus, mg 1214 (1218.2 ± 87.0) 1659 (1616.4 ± 104.1)

Potassium, mg (2803 ± 339) (2184 ± 290)1Per 2200 Kcal. Data in parentheses are analyzed values (mean ± SD)

Observed

0102030405060708090

100

0 5 10 15 20 25 30

Time (days)

Cal

cium

Ret

entio

n (%

) High MeatLow Meat

Modeled

0102030405060708090

100

0 5 10 15 20 25 30

Time (days)

Cal

cium

Ret

entio

n (%

)

High MeatLow Meat

Calcium Retention in Postmenopausal Women Consuming High and Low Meat Diets for 8 wk each

(Mean ±SEM)

Roughead, Johnson, Lykken & Hunt, (2003) J. Nutr. 133:1020

Diet: P = 0.09

High Meat Low Meat High Meat Low Meat

0

5

10

15

20

25

30

35

40

45Titratable AcidityDiet: P <0.0001Diet*Time: P <0.05

AmmoniumDiet: P <0.0001

week3 5 8 3 5 8 3 5 8 3 5 8

Roughead, Johnson, Lykken & Hunt, (2003) J. Nutr. 133:1020

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Diet did not Affect Biomarkers of Bone Metabolism

Blood:• Tartarate-resistant acid phosphatase, Bone-

specific alkaline phosphatase, Osteocalcin, 25-OH Vitamin D

Urine:• Calcium, N-telopeptide

Roughead, Johnson, Lykken & Hunt, (2003) J. Nutr. 133:1020

Protein Intake

Sulfur Amino Acid Intake

Osteoclastic activity Bone Resorption

Renal Net Acid Excretion

Glomerular Filtration Rate

Bone Mineral Density

Renal Reabsorption of Calcium

Bone Resorption

Bone Mineral Density

Net Endogenous Acid

⌧

⌧

⌧

⌧

Plantvs.

AnimalProteins?

ObjectivesIn healthy postmenopausal women, determine if daily substitution of 25 g of soy protein for meat protein1) improves calcium retention2) affects biomarkers of bone metabolism

Diet Composition1

CONTROL SOY

Meat (beef, chicken, pork), gMeat (beef, chicken, pork), g 170 55Soy Protein Isolate, g* --- 30Protein, % of energy 15 16

g/kg bw 1.32 1.33Fat, % of energy 30 30Carbohydrate, % of energy 55 55Calcium, mg 690 (653 ± 40) 746 (740 ±27)Phosphorus, mg 1505 (1563 ± 207) 1533 (1601 ± 178)Phytate, mg 1673 21501Per 2200 Kcal. Data in parentheses are analyzed values (mean ± SD)*Soy protein: 2.28 mg *Soy protein: 2.28 mg aglycone/gaglycone/g proteinprotein

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30Time (day)

0

10

20

30

40

50

60

70

80

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100

0 5 10 15 20 25 30Time (day)

CONTROL

SOY

Observed Modeled

Diet: NS (n=12)

Consuming 25 g of Soy Protein Substituted for Meat Protein for 8 wkConsuming 25 g of Soy Protein Substituted for Meat Protein for 8 wk did not Affect Calcium Retention in Postmenopausal Women did not Affect Calcium Retention in Postmenopausal Women

(Mean + SE)(Mean + SE)

Cal

cium

Ret

entio

n (%

of d

ose)

Observed Modeled

Roughead et al (2005) J. Clin. End. Met., in press

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1

10

100

0 5 10 15 20 25 30Time, days

Initial Absorption

Soy: 27.0 + 7.0Control: 26.1 + 7.0

NS

CONTROL SOY CONTROL SOY

0

10

20

30

40

50

60

week3 5 7 3 5 7 3 5 7 3 5 7

Ammonium(Diet: NS)

Titratable Acidity(Diet: P = 0.03)

Roughead et al (2005) J. Clin. End. Met., in press

The bottom line…

No evidence of adverse effects from animal protein intake on calcium metabolism even when calcium intake is low

What about whenCa intake is high?

0 1 2 3 7

Urine BloodUrine

5

Urine Urine BloodUrine

BloodUrine

Whole body Counting(28 days)

Controlled Diets(HC:1500 mg/d or LC: 600 mg/d)

47Ca

Note: Participant consumed either a Low or High Protein diet for 7 weeks; after a 2-wk washout, consumed the alternate diet for 7 weeks.

n=27

wk 1 wk 2 wk 3 wk 5 wk 70

20

40

60

80

100

120 HCHPHCLPLCHPLCLP

Ca: NS Pro: P < 0.0001Ca x Pro: NS

A high protein diet increased total renal acid excretion regardless of calcium intake

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wk 1 wk 2 wk 3 wk 5 wk 750

100

150

200

250HCHPHCLPLCHPLCLP

Ca: P < 0.0001Pro: P < 0.01Ca x Pro: NS

Both dietary calcium and protein independentlyinfluenced urinary calcium loss

0102030405060708090

100

0 5 10 15 20 25 30Time (days)

High Ca, High ProteinHigh Ca, Low ProteinLow Ca, High ProteinLow Ca, Low Protein

Calcium: P < 0.001Protein: P<0.02Ca x Pro: P <0.05

A high protein intake enhanced calcium retention from a low calcium diet

HCHP HCLP LCHP LCLP0

50

100

150

200

250

300

A high protein intake enhanced calcium bioavailabilityfrom a low calcium diet

Calcium: P < 0.001Protein: P<0.02Ca x Pro: P <0.05

HCHP HCLP LCHP LCLP0.0

5.0

10.0

15.0

Ca: NSPro: P < 0.001Ca x Pro: NS

A high protein intake reduced bone resorption as indicated by urinary Dpd excretion

HCHP HCLP LCHP LCLP0

50

100

150

200

A high protein diet increased serum IGF-1 by ~27%, regardless of calcium intake

Ca: NSPro: P< 0.01Ca x P: NS

ng/m

L

20% Meat

0 1 2 3 7 8 9 10 13 140

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90

100

Day

10% Meat20% Soy10% Soy

In ovx rats, the efficiency of calcium retention was highest when protein intake was high (especially from animal protein)

Source: P< 0.003Quantity: P <0.01Interaction: NS

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10% 20% 10% 20%0

200

400

600

800

1000

1200

Meat Soy

Source: NSQuantity: P = 0.02Interaction: NS

Serum IGF-1 was higher in the rats fed the high protein diets regardless of protein source

P Protein???

KMg

Milk Constituents

Ca

Vitamin D

Lactose

Dairy Protein and Bone?

• Isolated Casein and Lactalbumin have been shown to be calciuric

• Isolated Whey Protein may reduce bone resorption

Milk and Bone Health• Milk supplementation, 12 mo: increased spine and total

BMD in young girls (Chan, 1995)• Milk supplementation, 18 mo: increased serum IGF-I and

lumbar BMD and total BMC in young girls (Cadogan, 1997)• Milk supplementation, 3 y: prevented vertebral bone loss

in pre-meneopausal women (Baran, 1990)• High Ca milk supplementation 24 mo: decreased bone

loss in femoral neck, lumbar spine and total hip in postmenopausal women (Chee, 2003)

• Milk Supplementation 12 wk: increased serum IGF-I and decreased urinary N-telopeptides in older men and women (Heaney, 1999)

Protein Intake

Sulfur Amino Acid Intake

Acid Excretion

Increased urine Ca loss

Bone Mineral Density

Bone Resorption

↓ Calcium Retention

√

√

X, ?

X

X

↓

Bone Mineral Density

Serum IGF-1

?

?

Future Directions

Carefully controlled feeding trials are needed to assess:

• Effects of dairy proteins provided as food (milk, cheese, yogurt, etc) on calcium retention and bone health

• Effects of dairy proteins per se on calcium retention and bone health

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“As we analyze a thing into its parts and properties, we tend to magnify these, to exaggerate their apparent independence and to hide from ourselves the essential integrity and individuality for the composite whole. We may study them apart but it is a concession to our weakness and the narrow outlook of our minds.”

(D’Arcy Thompson, 1942)

Acknowledgments- Janet Hunt, PhD, RD - Bonnie Hoverson, RD- Glenn Lykken, PhD - Debbie Krause, RD - LuAnn Johnson - Brenda Hanson, RD- Carol Zito - Angela Scheett, RD- Jennifer Hanson - Emily Neilsen, RN• Jackie Nelson• Aldrin Lafferty• Study Volunteers• Primarily supported by USDA• Partial support: National Cattlemen’s Beef Association,

North Dakota Beef Commission• Solea Company (soy protein isolate)