Eric Orwoll, Marsha Tom

Sanchez,

ABSTRACT mineral

The

10 animals/group)

=

fed with induced

Daniel

Bikle,

on

understand specifically examined

of dietary

and

a protein-replete a profound

Lenka Stribrska, and Hongfei Li

are uncharacterized. We studied in protein (5%) for 4, 6, and 8 wks

effects

and bone metabolism rats fed a diet low

growing (n

Ware, Andon,

Mark

protein

compared

them

(18%) diet. hypocalciuria

restriction

with

animals

The low-protein that persisted

diet for

as well

were

absorption

mineral

pair-

were not affected D concentrations

calcium

lower

in the

protein,

bone

Male

MA)

relationship

meostasis has spite convincing result

in renal

calcium

balance dietary protein have suggested skeletal

protein

been the subject evidence that

calcium

on

between

loss

nutrition

and

skeletal

ho-

of considerable speculation. increases in dietary protein and

the

development

Deintake

of a negative

(I, 2), the skeletal implications of an excess in remain unclear. Whereas some investigations that high protein intakes have a negative impact

integrity

(3-5),

others

indicate

that

protein

effects genesis

variety

of situations

(12)

malnutrition,

difficult have

on bone. Protein of the osteopenia associated but

to establish

the

been concomitantly

aging

deficiency

(1 5-17)

19). Finally,

(14)

and

have

of insulin-like

which

might

314

adversely

(9-1

1) and

of protein studies.

impact

has been

growth affect

Protein

metabolism

associated

skeletal

1 (IGFhealth. Am

mass

in growing

rats.

methods

Fischer 344 rats (Charles River at 6 wk of age were acclimatized

Laboratory,

Wilmington,

to the experimental

sit-

group

was

The

switched

major other

to a low-protein

components regards

profiles)

the

derived

from

(vitamin,

diets

were

casein

(as dibasic

were designed protein content. ensure

of the

to

energy

mineral,

the

same.

hydrolysate

calcium

and

(5%

protein).

are shown in Table 1; fatty acid, and amino acid The protein in each diet was diets

and

each

phosphate)

be identical Dextrose was

equality,

formulation

two

diet

contained

and 0.79

with added

g P/L.

1.02

The

g

diets

the exception of the total to the low-protein diet to

an additional

0.021

g (2.6%)

P/L

in

the control diet was present as a result of the additional protein content. There was no difference in the pH of the diets. The animals receiving the low-protein diet were fed ad libitum, and the control the low-protein

rats were pair-fed group. Specifically,

to match the volume

the food intake ofdiet consumed

of

diet offered. Subgroups of low-protein-diet and control rats (10 in each subgroup) were killed at 4, 6, and 10 wk after the start of the diet. On the day before the rats were killed, 24-h urine were

collected

while

they

were

housed

in metabolic

is intake

(13). dietary

on bone

factor

bone

we on

nutrients

with BMD in children has been concern that in protein

and

study

specimens

animal

nutrition

of important

in these

a negative

concentrations in turn

role

alterations

deprivation

has been implicated been observed in a

human

a variety

deficient

may protein

with specific

because

has been positively associated ilarly, in elderly people there tein

deficiency that has

on the skeleton

by each animal in the low-protein group was assessed daily and, hence, determined the volume ofdiet offered the control animals on the next day. Control animals routinely consumed all of the

intake

has no relationship to skeletal mass or may actually be positively related to bone mineral density (BMD) (6-8). Conversely, protein deprivation has also been reported to have adverse in the

deficiency

uation for 7 d. During this period they were fed a control (18% protein, Bio-Serve, Frenchtown, NJ)liquid formula diet. Animals were then divided into two groups (30 animals/group) and one

Ca/L

Introduction The

of protein

and

and

Chronic

in

Calciuria,

metabolism

Materials

low-protein animals. Skeletal dimensions were reduced in the protein-deprived rats but there were no significant differences in bone mineral content between control and low-protein animals at 4, 6, and 8 wks. Hence, dietary protein deprivation resulted in slower growth but bone mineral density was maintained when there was a marked reduction in urinary calcium excretion. Am J Clin Nuir l992;56:3 14-9. KEY WORDS

the effects

on the process of mineralization during growth, the acute and chronic effects of protein restriction

rapidly 8 wk.

calcium and phosphorus concentrations but serum total and free 25-dihydroxyvitamin

Serum

as gastrointestinal

on mineral metabolism

J Clin

Simprowith

mass with

(18, lower

1) (20, 21), To Nuir

better 1992;56:314-9.

I From the Portland Veterans Administration Medical Center and the Oregon Health Sciences University, Portland, OR; the San Francisco Veterans Administration Medical Center and the University of California, San Francisco; the Norland Corporation, Ft Atkinson, WI; and The

Miami

Valley

Address

Laboratory,

Printed

reprint

Cincinnati.

to ES Orwoll, Portland (1 1 1), P0 Box 1034, Portland, OR 97207. Received October 28, 1991. Accepted for publication January 28, 1992. 2

requests

in USA.

© 1992

American

Society

VA Medical

for Clinical

Service

Nutrition

Downloaded from https://academic.oup.com/ajcn/article-abstract/56/2/314/4715367 by University of Rhode Island user on 05 December 2018

Effects of dietary protein deficiency and bone mineral density1’2

PROTEIN

TABLE Diet

INTAKE

AND

1

MINERAL

resulted

composition

in greater

equivalent Control

taming

5

735 1995 1470 1.15 0.61

210 2520 1470 0.56 0.53

1.02 0.79 0.13 10

1.02 0.79 0.13 10 1800

albumin,

trations

of calcium,

was stored

was at -20

were collected, Seattle)

cleaned

apparatus,

ofsoft

defatted

measured

in a Soxhlet

(length),

and

Bone

for

study

San

ofpair-fed animals (male, Fischer 344, Charles were studied after consuming liquid diets for

formulation

were initially group was

for an additional

were collected Intestinal

under

cakium

A third group River Laboratory)

on a control diet for to the low-protein 24-h

urine

specimens

cages.

of 20 pair-fed rats (male, were fed either control

group)

a 24-h urine

as above

for 4 wk.

specimen

was then

body radioisotope retention was added to 3-mL aliquots

density

Fischer 344, Charles or low-protein diets

Three

days

was collected.

estimated technique

with (22,

before

the

rats

Gastrointestinal the use ofa 23).

47Ca (370

wholekBg)

gavage. The radioactivity in the animals was determined (within 2 h after gavage) by whole-body gamma counting, and access and water was resumed. Whole-body radioactivity was again determined 48 h later (with all counts corrected for background and decay) for calculation of the percent retention of to diet

radioactivity. The transit time of liquid diets in rats is short (< 48 h). This method gives a relative rather than an absolute of calcium absorption related to tests of true

but it has been absorption (24).

reported to be Rats were then

was collected and frozen. To determine whether or low-protein diet had direct (immediate) effects on calcium absorption, 47Ca was preequilibrated with aliquots of the two diets, which were then used to assess absorption in groups killed

and

serum

the control

of rats (seven animals in each (Purina, St Louis). Absorption with that from the low-protein

than

that

from

Capistrano,

sodium

were

hormone

con-

immunoassay

CA),

serum

25-dihy-

measurements femoral

and tibial

dition,

densities

absorptiometry

Atkinson, WI). Each results were averaged. whole-femur (26).

taken

density

by using

Corporation,

Fort

was determined

(model

Bone

were measured

(Norland

specimen was assessed five times and the The CV of these measures is 5%. In ad-

x-ray absorptiometry

mineral

XR-26; content

as the average

Norland) (BMC)

scanning

of five scans

by using dual-energy as described preand BMD (g/cm2)

sample. of 3.0 mm/s and a point resolution ofO.5 mm. Repeated measures of femur specimens revealed a precision (CV) of 1.7% for BMC and 1.5% for BMD. Comparisons between groups were performed by using unpaired t tests. This protocol was reviewed and approved by the Animal Care Committee, Portland Veterans Administration Medical

ofcontrol or low-protein diets and allowed to equilibrate for 24 h (4 #{176}C) before administration. After being deprived of diet for 16 h and water for 2 h, rats received one of the two 47Ca-containing diets by intragastric

estimate closely

and

parathyroid

concen-

was

performed

made

with

on each

a scan

speed

Center.

absorption

(10 in each

absorption

5 d. Daily

oil in metabolic

were killed, calcium

placed switched

Juan

phos-

urine

D [l,25-(OH)2D] concentrations by receptorassay (Incstar, Stillwater, MN), and the percent free 1,25concentrations by previously published methods (25).

Rectilinear

the

creatinine,

by amino-terminal

viously

8 d. Twelve animals 3 d and then half

phosphorus,

measured

Institute,

calcium, and

were

were A second group River Laboratory)

creatinine,

centrations

(OH)2D

analysis.

Acute

an

phosphate-con-

phosphatase, Serum

binding

(VWR, stored

± 3%) from

droxyvitamin

time the rats were killed and analysis. Tibia and femurs

tissue,

weighed,

alkaline

by autoanalyzer.

(Nichols

collected at the #{176}C for subsequent

of sodium,

and

determined

Midshaft Serum

(18

calcium

solution.

concentrations

phorus,

single-photon

cages.

that

dibasic

determinations

Serum

1800

than

of

47Ca in an aqueous

Biochemical

18

absorption

concentration

the control

group) from

previously fed a stock diet the two diets was similar, diet [37 ± 5% (SD)] slightly greater

diet

(30

± 4%).

Both

liquid

diets

Results Changes in body weight and in serum and urine biochemical variables after 4, 6, and 8 wk of control or low-protein feeding are shown in Table 2. Body weight was lower at all time points in the animals

protein

consuming was

the

deprivation

growth, although was no obvious

growth

low-protein

clearly

sufficient

continued

at a slower

lower

in the

equaled those concentrations imals

protein-deprived

of the control were slightly

at 8 wk.

reduced

of kwashiorkor

Hence,

to impair rate

in the

Serum

total

protein-deprived

the percent free l,25-(OH)D toward lower concentrations tein diet (P > 0.05). Whereas

serum

creatinine

(edema,

the

optimal and

there

hair loss). Serum albumin concentrations were lower in the proteindeprived rats after 4 and 6 wk but were not different from the control rats at 8 wk. Apparently reflecting the difference in albumin concentrations, total serum calcium concentrations were also

evidence

formula.

groups

group higher

at 4 and

6 wk

but

at 8 wk. Serum phosphorus in the protein-deprived an-

l,25-(OH)2D rats

apathy,

concentrations at 8 wk.

concentrations in the group concentrations

Measurement

were of

also revealed a trend receiving the low-prowere

not altered

by

the low-protein diet, urinary creatinine excretion was considerably reduced. More striking, however, was the dramatic reduction ofurinary calcium in the protein-deprived animals. This was true at all time points but particularly at 4 wk, and the reduction in calcium excretion was accompanied by an increase

Downloaded from https://academic.oup.com/ajcn/article-abstract/56/2/314/4715367 by University of Rhode Island user on 05 December 2018

Protein (%) Energy content (kJ/L) Protein Carbohydrate Fat Methionine (gIL) Cystine (gIL) Mineral content (gIL) Calcium Phosphorus Magnesium Vitamin D (ig/L) Vitamin A (zg/L)

Low protein

315

METABOLISM

ORWOLL

316 TABLE 2 Body weight

and serum

and urine

biochemical

variables

after

ET AL

4. 6, and 8 wk ofthe

control

4wk

± 9

16.0

± 0.071 2.81 ± 0.19 53±9

28 ±

1

±

191 ±

2.57

33 ± 2

(ng/L) D

C

122 ± lOt

2.87 ± 0.22 2.58 ± 0.42 53±9

16.0

±

LP 16

161 ±

35 ±

2

141

262 ±

2.72

2.79 ± 0.07 2.91 ± 0.23 53±9

It

C

± 0.lt 2.71 ± 0.23 53±9

1

29 ±

-

-

-

-

-

-

-

-

-

-

LP 12

223 ± 2lt

2.79 ± 0.02 2.29 ± 0.19 53±9

It

33 ±

2.77 2.45

.07 .l3t

± ±

53±9

1

31 ± 2

-

-

190 ± 36

134

± 38

D 0.18

±

0.05

0.14

±

0.07

1.25 0.04 0.13

±

0.32 0.01 0.13

0.32 .01 3.04

±

0.12

±

0.006t

±

0.55t

530 ± 7 1

442

(24 h)

Calcium (mmol/L) Calcium: chromium chloride Phosphorus (mmol/L) Creatinine (umol/L) Volume (L) *

LP

8wk

.‘

tt*

±

SD:

ii

=

volving

different

excretion.

a short

increased

bicarbonate animals = 0.05)

±

0.42 0.03 2.26

0.004

±

± ± ±

0.2t 0.It 0.58t

4.59 0.16 0.13

265 ± 27t

± 44

0.03

group):

effect (Figs on the

1-3)

in the controls remained lower

became