Protein deficiency: its effects on body temperature in health and disease states14 Hoffman-Goetz,5

Ph.D.

ABSTRACT

body

and

Matthew

is known

Little

temperature

a low-protein

during diet

and

J. Kluger,6

health

about

the

Ph.D.

effects

ofprotein

malnutrition

and disease. To investigate their body temperatures.

recorded

on the

this area, There

ability

we placed

were

no

to regulate

young

rabbits

differences

on

between

the

and control animals concerning their abilities to maintain constant body temperatures during exposure to low (5 C, 10 C) and thermoneutral ambient temperature (20 C). In a warm ambient temperature (30 C) the protein-deprived animals were actually better able to maintain a lower body temperature. Injections with heat killed bacteria led to little or no fever in the protein-deprived group. However, intravenous injections of endogenous pyrogen, a protein mediator of fever, resulted in fevers vutually identical to that attained in control animals. These data indicate that the attenuated febrile response to bacterial injection during protein deprivation protein-deprived

may central

be due

to a diminished

nervous

system

production

sensitivity

of endogenous

It is a widespread clinical impression that protein malnourished children have difficulty regulating their body temperatures (1). There have been reports of hypothermia in the cold (2),

and

this

has

conservation.

been

attributed

to

chronic

of the

bacterial

infections

etc.)

A

careful literature search has failed to reveal an animal model that has been used to investigate the effects of protein malnutrition on the ability to regulate body temperature. In an attempt to simulate simple protein deficiency and to study the thermoregulatory responses associated with protein deficiency, young rabbits fed a low-protein diet were monitored for thermoregulatory responses to changes in ambient temperature, to injections with killed bacteria, and to injections with endogenous pyrogen, a protein mediator of fever. We report that whereas protein deprivation has little effect on the ability of rabbits to regulate their body temperature during exposure to cold stress, protein deprivation abolishes or severely attenuates the febrile response after inoculation with dead bacteria. The American

Journal

of Clinical

Nutrition

Am.

and

J. Clin.

Intravenous gen, however,

Nuir.

to some 1423-1427.

injections produce

protein-deprived

parable

not 32:

in the

1979.

of endogenous fever responses

rabbits

magnitude

alteration

which

to fevers

pyroin the

are

of

com-

in the controls.

calorie

problems with interpreting these types of clinical data is that, for obvious reasons, these have been uncontrolled experiments. Seldom were the patients simply protein deprived. Generally associated with this protein malnutrition were a host of related ailments (e.g., vitamin deficiencies,

One

pyrogen,

to pyrogens.

32: JULY

Materials

and methods

Juvenile

male New Zealand white rabbits (Oryctolaweighing between 2.25 to 3.0 kg. were assigned to one of two dietary groups. Group

cuniculus)

gus

randomly 1 included

healthy

ston-Purina

Lab

of

10.0%

tamin

fat,

animals

Rabbit

70.15%

fed

Chow

a low

protein diet (Ralthat consisted 4.5% protein, a vi-

no. 5767)

carbohydrate,

and mineral mixture, and Group 2 included healthy

nonnutritive

fiber

to

100%. animals pair-fed a standard pelleted lab rabbit chow (Ralston-Purina Chow no. 5755). Water was given ad libitum. The rabbits received the low-protein diet until the serum albumin level fell to below 2.5 g/lOO ml which required 7 to 8

weeks.

The animals

steel cages photoperiod.

and

were housed

were

individually on

maintained

in stainless

a 12-hr

light/dark

1From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109. 2Supported in part by Grant AI-l3878 from the National

Institutes

of Health,

Bethesda,

3Address reprint requests Goetz, Nutrition/Metabolism England Deaconess Hospital, Massachusetts 02215.

‘The the

animals

were

and

and regulations of the National Research Fellow.

handled

rules

of the

Research

Maryland.

to: Dr.

Laurie

Laboratory-C.R.I., 194 Pilgrim Road,

in strict

accordance

Animal

Resources

HoffmanNew

Boston, with Board

Council.

6 Associate

Professor

of Physi-

ology.

1979, pp. 1423-1427.

Printed

in

U.S.A.

1423

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Laurie

HOFFMAN-GOETZ

1424

Intraabdominal

temperatures thermocouples.

copper-constantan

thermocouples, were threaded anesthetized

covered beneath rabbits

HCI,

measured Copper-constantan

using

with sterile polyethylene tubing, the middorsal skin of lightly

(1 ml

aceproma.zine,

i.m.

and

1 ml

s.c.)

and inserted into the peritoneum using sterile stainless steel trochars. The thermocouples were then sutured to the dorsum, and the animals were placed in modified neck restrainers (3) to prevent thermocouple displacement. The abdominal temperature of each animal was recorded each 30 sec on a Honeywell Electronik 1 12 multipoint recorder. During those experiments in which we tested the rabbits’ ability to regulate their body temperatures at

different ambient temperatures, groups were placed in a sealed environmental maintained

any

The

given

ambient

of four chamber

temperature

±

rabbits which

1.0 C.

bacterium Pasturella multocida was selected because it is known to be a fever producing pathogen to rabbits (4, 5). Bacteria from stock cultures (American Type Culture no. 7228) were grown on sheep blood

Gram-negative

agar

pyrogen-free

plates,

incubated

48 hr at 37 C, suspended

saline, centrifuged 0.9% saline. Bacterial

twice,

in

and resuspended

in sterile concentrations were determined with a Beckman DB Spectrophotometer, based upon reference curves determined from McFarland Barium Sulfate turbidity standards. Bacteria were heat killed by autoclaving at 121 C for 30 min.

Rabbit

endogenous based

pyrogen

was obtained

from

toneal exudates (6). The technique

on the method of Bornstein was modified in two respects:

shellfish

solution

glycogen

peritonitis not dialyzed

in rabbits, against

endogenous granulocytes

pyrogen from

was

and the a normal

used

are expressed which

the

to induce

endogenous salt solution.

periet al. a 0.2%

acute

pyrogen was All doses of

as concentration

endogenous

pyrogen

of was

obtained.

KLUGER

ature

(20

lower rabbits

body temperature during exposure

C:

bly reflects

these

F

body weights despite pair

3). The average 6-hr fever in the low-protein group given 1 ml of 1 x iO bacteria per

milliliter was 0.14 C compared to the control fever of 0.71 C (F < 0.01, n = 7). The mean 6-hr fever response in the low-protein rabbits given a 10-fold increase in bacteria (1 ml of 1 x 108 bacteria per milliliter) was 0.33 C, whereas the control group had an average fever of 0.77 C (F < 0.005, n = 22). At the highest dose of bacteria ( 1 x l0) the lowprotein rabbits again showed a diminished 6hr fever (0.38 C) compared with the control rabbits (0.82 C) (P < 0.005, n = 9). These

of rabbits. and day.

to challenge

Figure

=

of

with

fever

1 shows

the

results

of body

weight

19).

There were no differences in the responses of the rabbits fed the low diet and those fed the control diet exposure to severe cold stress (5 C: n = 4), moderate cold stress (10 C: n = 11) and thermoneutral ambient

thermal protein during F> F>

0.3, 0.3,

temper-

protein-deprived

fever

heat-killed

believed (e.g.,

Gram-positive tion

that

a diminished

It is currently tors

and serum albumin changes over 8 weeks in the control and low-protein diet groups. Mean body weight at the termination of the feeding regime was significantly different in the two groups (P < 0.005, n = 33). Serum albumin levels in the low-protein rabbits fell from an average of 4.36 to 2.28 g/lOO ml over the 8-week feeding regimens (P < 0.005, n

of

in-

the two dietary groups Data obtained from cooling curves (n = 5) confirm the faster and greater heat loss of the low-protein animals at any given ambient temperature. Intravenous injections (1 ml of 1 x l0, 1 x 108, and 1 x 10k, bacteria per milliliter) with heat killed F. multocida led to little or no fever in the low-protein diet fed rabbits (Fig.

demonstrate

Results

capacity

of decreased

between feeding.

have

paired

heat-losing

sulation (decreased subcutaneous fat, pronounced hair loss). This decreased insulation also accounts for the significant difference in

rabbits

for each hour using t and

7) (Fig. 2). The of the low-protein to heat stress proba=

as a function

results

calculated analyzed

n

the greater

animals

Mean hourly and daily body temperatures were obtained by calculating the combined means for each group Standard errors were Data were statistically t tests.

0.3,

>

that

various

Gram-negative

bacteria)

of endogenous

in response

bacteria.

induce

pyrogen,

activabacteria,

the

a low

producmolecular

weight protein produced by leukocytes, Kupffer cells (fixed tissue macrophages) and other physiologically active phagocytic cells. It is this endogenous pyrogen that circulates to the hypothalamus and tion in the thermoregulatory

induces the “set-point”

eleva(7).

Since these low-protein fed rabbits could regulate their body temperature in the cold, we suspected

that

central

nervous

system

control

of temperature regulation was not impaired and perhaps these animals were developing reduced fevers because of a diminished capacity for producing test this hypothesis,

endogenous we injected

protein

rabbits with endogenous 4 summarizes the fe-

pyrogen

and

control

(i.v.).

Figure

pyrogen. both

To low-

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lidocaine

were

AND

PROTEIN

1425

DEFICIENCY

DAYS

C,

w b) 0

I

7

$4

21

28

35

42

49

56

DAYS FIG. I. a, Serum albumin concentrations (g/lOO ml) changes for 8 weeks in rabbits fed a low-protein diet and in rabbits fed a control diet. All values indicate the sample means ± SEM. b, changes in total body weight over 8 weeks in rabbits fed a low-protein diet and in rabbits fed a control diet. All values are sample means ± SEM.

brile characteristics of the low-protein fed rabbits and the control rabbits for three doses of endogenous pyrogen. There were no significant differences between the febrile responses in the low-protein and control rabbits at the three doses of endogenous pyrogen. These results demonstrate that protein deprived rabbits do develop fever when endogenous pyrogen is administered intravenously. Discussion This study demonstrates that protein deprivation in young rabbits attenuates the ability to generate a fever in response to injection

with

killed

Gram-negative

bacteria,

not diminish the ability to regulate perature under cold stress. Several tions might be advanced to explain

but does body temexplanathe failure

to develop fever during chronic protein deprivation. Three important possibilities are: 1) Impaired ability to produce sufficient heat to raise body temperature. This is possible, but unlikely given the ability of the protein deprived rabbits to maintain constant body temperature in the cold. 2) Defective or decreased sensitivity of the hypothalamic areas sensitive to pyrogens. hypothalamus ability of the

Functional insensitivity is also unlikely given protein deprived rabbits

of the both the to nor-

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a)

HOFFMAN-GOETZ

1426

AND

KLUGER

CONTROL LOW

-

PROTEIN

I

40.5

T0’

L-T-

30C

39.5

I-.

38.5 405 T0:20’C

i 40.5 T0’

IOC 1.5

&

Control

#{149} Low

39.5

I

38.5

(n’9) prot.sn

zIOS

(n.13)

P multocido

,

40.5 T0’

5C

39.5

F’

38.5 I

LIGHT

6

ON

8

12

24

HOURS FIG.

2.

moneutrality, protein diet

Body

under

34

2

cold

stress,

ther-

5 6 HOURS

and heat stress in rabbits fed the lowthe control diet. Each plot represents an 24-hr cycle at any given ambient temperarepresent sample mean ± SEM.

7

8

9

Control

(n.3)

Values

#{149} Low piotsm 1*10’

mally

10

and

independent ture.

temperature

thermoregulate

temperatures,

and

in

the

stressful

ability

ambient

of the

protein

deprived rabbits to respond with fevers when injected with endogenous pyrogen. 3) Dccrease or absence of production of endogenous pyrogen, a protein mediator of fever. The results obtained in this study support the last interpretation. We believe that the failure to produce adequate endogenous pyrogen by protein deprived rabbits during infection may be the result of either; 1) a depressed leuko-

cyte count, specifically that class of leukocytes involved in endogenous pyrogen production (e.g., granulocytes, monocytes) or 2) a depressed biochemical synthesis of endogenous pyrogen. These diminished endogenous

parameters pyrogen

involved production

a necessary of the febrile

prerequisite response.

U I-

0

I

234 HOURS

FIG. 3. Fever response in low-protein and control fed rabbits given 1 ml of 1 x l0, 1 x l0, and 1 x 10#{176} P. multocida per milliter. All injections were given intravenously.

perature

Values

represent

the

pooled

changes

in

tem-

per sample.

in

in protein-deprived rabbits are currently being investigated. The ability to produce endogenous pyrogen is, then, functioning

(n.4)

fmultoc.do

for the Indeed,

various

studies

have

demonstrated

that

leu-

kocyte functions, other than those involved in the production of endogenous pyrogen, are impaired during protein malnutrition (e.g., Reference 8). Although much attention has recently been

Downloaded from https://academic.oup.com/ajcn/article-abstract/32/7/1423/4692216 by University of Otago user on 17 December 2018

U

PROTEIN

1.5 . 40

a

I0

6

LOW

A

CONTROL

DEFICIENCY

1427

the host

PROTEIN

are

growth.

LEUKOCYTES

necessary

for optimal

An endogenous

protein

bacterial

mediator,

bi-

I.0

ologically pyrogen

(14) indistinguishable and released

triggers

the

from

immune

the sequestering of serum and thereby decreasing free

I

0

endogenous host leuko-

reaction

leading

iron to the iron available

to

liver for

1.5 701 106

bacterial

LEUKOCYTES

growth.

It is, therefore,

quite

likely

1.0 6

0.5

N.

that thehigh diminished pyrogen production results tively not during only serum in chronic iron aendogenous reduced levels proteinasfever, well. deprivation butHence, rela-

N.6

a primary defect in the ability to produce endogenous pyrogen as a result of protein deprivation may play a decisive role in hostbacteria interactions. El

0 1.5 4OiIctLEUK0cVTES .

N.?

References

N.5

‘20

40

60

100

80

120

140

TIME (minutss) FIG. 4. Fever characteristics of low-protein and control rabbits given varying doses of endogenous pyrogen. The doses of endogenous pyrogen consisted of 40 x 108, 70 x 106, and 140 x 106 granulocytes. All values represent the pooled sample changes in temperature.

focused on fever during

the potential survival value of infection (9, 10), the specific

causal

relationships

vival

in mammals

nonspecific eficial to

attenuated malnutrition of the host’s

between

are not known.

host response survival, then

febrile might ability

fever

to infection, the presence

response

during

and

a

is benof an

protein

result in an impairment to combat infection.

Second,

it is also

likely

that

1972. BRENTON,

Influence of malnutrition of children. Brit. Med.

on the J. 1: 331,

R. E. BROWN AND B. A. WHARTON. in kwasbiorkor. Lancet 1: 410, 1967. 3. HAMPTON, G. R., W. V. Sp AND C. J. ANDansEN. Long term rabbit restraint-a simple method. Lab. Animal Sci. 23: 590, 1973. 4. FLATr, R. E. Bacterial diseases. In: The Biology of

2.

D.,

Hypothermia

the

Rabbit,

Laboratory

edited

R. E. Flatt

and

A. L. Kraus.

Press,

pp.

193-236.

l974,

5. H0FFMAN-G0ETz, protein deficiency Federation Proc. 6. B0ENsTaIN, D. WOOD. Studies

by

New

S. H. Weisbroth,

York:

Academic

L., AND M. J. KLUGER. Effect and bacteria on body temperature. 37: 930, 1978. L., C. BRzrminERG

ir

on

of fever

the

pathogenesis

B.

of

W.

XI.

Quantitative features of the febrile response to leucocytic pyrogen. J. Exptl. Med. 1 17: 349, 1963.

The reduced production of endogenous pyrogen by protein deprived animals has at least two other important implications for host survival during bacterial infection. First, fever is the most widespread clinical manifestation of infection (1 1) and one of the most consistently utilized diagnostic markers of infection for the physician. Failure to mount a fever during the early stages of bacterial infections in protein malnourished children would necessitate more elaborate clinical tests to determine the nature of the illness. Lack of fever might not indicate lack of disease in the host compromised by protein deficiency syndromes.

body

sur-

If fever,

0. G. temperature

I. BROOKE,

7. SNnu., E. S., AND E. fever. In: The Biological edited

E.

E.

Bittar

Basis and

The mechanisms of of Medicine, Vol. 2,

N.

Bittar.

New

York:

Press, 1968, pp. 397-419. S. D., AND K. SCHOPPER.

DOUGLAS, The phagocyte in protein-calorie malnutrition-a review. In: Malnutrition and the Immune Response, edited by R. Suskind. New York: Raven Press, 1977, pp. 231-243. 9. KLUGER, M. J., D. RINGLER AND M. ANvaR. Fever and survival. Science 188: 166, 1975. 10. KLUGER, M. J. The evolution and adaptive value of fever. Am. Sci. 66: 38, 1978. 11. ATKINS, E., AND P. BODEL. Fever. New Engl. J. Med.

8.

286: 12. 13.

this

reduced production of endogenous pyrogen results in many other immunological defects as well. Weinberg (12), Beisel(l3), and others have shown that high levels of serum iron in

by

Academic

ATKINS.

27,

E. Iron

disease.

Science

BEISEL,

W.

responses 1977. 14.

1972.

WEINBERG,

184:

and

susceptibility

952,

R. Magnitude Am.

to infection.

MERRIMAN,

KAMPSCHMIDT.

C.

R.,

L.

to infectious

1974.

A.

Comparison

and leukocytic endogenous Exptl. Biol. Med. 154: 224,

of the host nutritional J. Clin. Nutr. 30: 1236, Puuii AND R. F. of leukocytic pyrogen mediator. Proc. Soc. 1977.

Downloaded from https://academic.oup.com/ajcn/article-abstract/32/7/1423/4692216 by University of Otago user on 17 December 2018

cytes,

‘10.5