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Carcinogenesis vol.8 no.7 pp.871-873, 1987
COMMENTARY
Nutrient density: an important and useful tool for laboratory
animal studies
Harold L.Newmark
Sloan-Kettering Institute for Cancer Research, Laboratory for Gastrointestinal
Cancer Research, 1275 York Avenue, New York, NY 10021, USA
Dietary considerations in animal studies
Nutritional factors are of growing interest in relation to human
diseases, particularly those that are seemingly related to longterm metabolic derangements such as those which occur in
cancer. For example, epidemiologic studies in humans have
revealed a few correlations between dietary habits and increased risk of cancer. Thus a diet rich in fat and meat, but low in
cereals, fiber, fruits and yellow and green vegetables correlates
with increased risk for colorectal and mammary cancer in humans
(1). Studies of this type seem to identify one or more dietary
factors that are associated with an increased risk for certain kinds
of cancer.
Once identified by epidemiologic studies, investigators often
wish to study these suspected dietary factors in laboratory
animals. This poses problems, long known to the early pioneers
in nutrition research, of translating nutritional requirements and
nutrient interactions from humans to the species used in the
laboratory studies. A few of these problems, and methods of dealing with them, are discussed below.
Metabolic rate differences between species
Even adult animals of different species may have large differences
in metabolic rate. This is evident by the calories of energy required per kg of body weight to maintain body weight while
engaged in 'normal' activities, which can vary from 80 kcal/day
for a 200-g rat (400 kcal/kg), to 2400 kcal/day for a 60-kg human
(40 kcal/kg body wt). This illustrates an ~ 10-fold difference in
energy requirement between an adult rat and human when expressed on a body weight basis.
Age differences
In all species, nutrient requirements for young animals are greater
than those for adults due to the requirements for production of
new tissues for growth. In addition, younger animals usually have
a higher metabolic rate than adults, further increasing energy and
nutrient requirements on a body weight basis.
Body surface area
Body surface area has been used as a means of calculating nutrient
intake and drug dosage for young animals (or humans) (2). The
metabolic rate of homothermal animals is more nearly propor© IRL Press Limited, Oxford, England
Nutrient density
This is probably the simplest and most useful method of comparing adequacy of nutrient requirements between species, and
between young and old animals. This method relates the quantities of all nutrients to the energy content of the diet, usually
expressed as mg or units of nutrient per kcal of diet. Increased
dietary intake of an animal due to higher metabolic rate, thus
requiring more energy for balance, then results in proprotionately
increased intake of all nutrients. This method is a common tool
in many areas of nutrition research and in domestic farm animal
production. Nutrient density is frequently used in calculations
and definition of nutrient requirements, particularly of nutrients
that are not extensively stored in mammals. The human US
Recommended Dietary Allowances for thiamin and riboflavin
nutrition requirements are defined in terms of mg per 1000 kcal
of diet (4). The concept of nutrient density has long been applied to protein intake, and it has been proposed as a basic concept for zinc as well (5). In animal nutrition, the energy (caloric)
intake can be expressed as 'gross energy', 'digestible energy'
or 'metabolizable energy' (6). For laboratory animals metabolizable energy is generally acceptable (4 kcal/g for protein and carbohydrate; 9 kcal/g for fat; 7 kcal/g for alcohol). Alternatively,
it may be sufficiently useful to use the approximate easily obtained values for gross energy, minus those fiber components
(cellulose, lignin) known not to be metabolized by the animals
used in the laboratory.
Relation of nutrient density to carcinogenesis
Applications of the concept of nutrient density of nutrients in
studies of carcinogenesis can be very useful. Laboratory animal
diets, particularly for rodents (mice and rats), are frequently
designed to be optimum for growth of young animals, and are
probably 'too rich in protein, vitamins and minerals for adult
animals, resulting in unnatural obesity in adults' (7). Obesity itself
may magnify carcinogenesis in laboratory studies (8). Laboratory
animal experiments with adult animals in carcinogenesis studies
should preferably adjust the levels of suspect key nutrients to
their density in the diet of the human population considered at
risk. Sometimes this is done, as with studies on high dietary fat
using 20—30% (by weight) of dietary fat in an experimental
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Nutritional deficiencies or imbalances are suspected contributory factors to several types of human cancers, and
perhaps other human diseases related to long-term metabolic
derangements. In order to study these more effectively in
laboratory animals, it is suggested that the laboratory diets
more closely mimic the nutrient density of suspect human
diets. Toxicology and carcinogenic data obtained in animal
study using diets based on human nutrient density might be
more readily applicable in relation to humans.
tional to the body surface area than to the weight, although not
strictly proportional (2,3). It has also been used as a means of
calculating nutrient intake and drug dosage for young animals
(or humans) (2). The metabolic rate of homothermal animals is
more nearly proportional to the body surface area than to the
weight, although not strictly proportional (2,3). It has also been
found that the metabolic rate, divided by 3/4 the power of the
body wt in kg, is independent of the body size. Under normal
conditions this quotient, from mice to cattle, averages 70 kcal
per 3/4 the power of kg (body wt) per day (2,3). This represents
another alternative in calculating inter-species nutrient requirement, but is complex and seldom used for comparison of nutrient
requirements between species.
H.L.Newmark
Table I. Daily calcium dietary intake
Species
Man (23-50)"
Woman (23-50)*
Mouseb
Rat0
Weight
70 kg
55 kg
20 g
200 g
kcal/day
2700
2200
8
80
Daily calcium11
RDA
Body wt
(mg/kg)
(mg)
mf{5CGf
800^500)*
10" (12/
10(^(120/
Diet
(mg/kcal)
11 (7) 0.30(0.19)
15 (9) 0.40(0.25)
500(600) 1.25(1.5)
500(600) 1.25(1.5)
"Data from recommended daily allowancs, RDA, 9th edn 1980 (4).
b
Mouse data based on an adult mouse of 20 g consuming 2 g/day of
AIN-76 rodent feed, containing 3.6 kcal/g and 0.5% calcium (9).
°Rat data based on an adult rat of 200 g consuming 20 g/day of AIN-76 rodent feed, calculated similarly to mouse (footnote b).
^The numbers in the columns headed by calcium are based on the RDA
values. The numers in parenthesis are based on estimates of actual intakes.
T h e estimate of actual intake is from ref. 19.
'Laboratory rodent feeds contain a minimum of 0.5% calcium (9).
However, many rodent feeds contain considerably more (up to 0.9%) (20).
The estimate of actual intake chosen here was 0.6%.
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tion in the US that has a calcium intake of - 2 0 0 - 2 5 0 mg/day
(4), representing a nutrient density of 0.1 mg/kcal, carcinogenic
studies in laboratory animals with feeds at 3.5 - 4 . 0 kcal/g of feed
should contain 0.04% calcium to mimic the human diet in terms
of calcium intake. This is about one-tenth the RDA level of 0.5%
of calcium in normal laboratory rodent feeds.
(ii) The human requirement for ascorbic acid has been
calculated by Pauling (14) based on a study in adult normal rats
which established a rate of biosynthesis of — 26 mg/day per kg
of body wt to normally saturate its tissues (15). Extrapolation
to human adults (60-70 kg) on a body weight basis suggested
to Pauling a requirement of up to 1.8—4.1 g/day (14). However,
calculated on a nutrient density basis, the rat biosynthesis of
26 mg/day of ascorbic acid per kg of body wt would be calculated
against the -400 kcal/day of dietary energy per kg of body wt
required by the rat, equivalent to - 0.065 mg/kcal (diet) per day.
Extrapolation of this value to estimates of human ascorbic acid
requirements based on nutrient density suggests values of
115 -175 mg of ascorbic acid for human diets ranging from 1800
to 2700 kcal per day. These calculations are close to the level
of intake of 100 mg/day which was experimentally found to maintain the maximum tissue pool size of ascorbic acid in humans,
indicating the utility of species extrapolation using the principle
of nutrient density (16).
(iii) In a recent study in female rats, Jacobson et al. (17) varied
the calcium intake of the diet from 1.5 mg/kcal (i.e. the rodent
RDA) down to 0.25 mg/kcal (i.e. equivalent to the human RDA)
and also 0.1 mg/kcal (equivalent to a low adult human intake
of 250 mg calcium on a 2500 kcal daily diet), on low fat (3%
by weight) and high fat (20% by weight, or 40% of calories)
diet. After 12 weeks on these diets, the mammary epithelial proliferation rate as measured by [3H]thymidine-labelling index was
several-fold higher in the rats on the lower calcium levels that
mimic the human nutrient density levels. This was the case within
both the low and high fat groups. In a parallel preliminary experiment with DMBA-induced mammary tumors, similar large
differences in tumor production were noted after feeding the diets
for 29 weeks. These results strongly suggest that utilization of
nutrient density can be a useful tool in studies of dietary modulation of mammary carcinogenesis. In another recent study in
female mice in another laboratory, Zhang et al. (18) have also
found that variation of dietary calcium significantly affects the
proliferation status of mammary glands measured by [3H]thymidine-labelling index. The labelling index for the terminal
ducts on high fat (corn oil) diets decreased from 14.1, 11.9 to
8.5, as the calcium levels were increased from 0.1% of diet
(nutrient density of -0.25 mg/kcal, equivalent to human RDA),
to 0.5% of diet ( - 1 . 2 5 mg/kcal), and to 1% of diet
(— 2.5 mg/kcal). Mitotic indices followed the same trend. These
studies, which mimic the human diet by controlling the fat and
calcium levels to mimic the human diets utilizing the principle
of nutrient density, by percent of total calories, or amount of
nutrient per kcal of diet, have demonstrated the implications of
dietary calcium as a modulator of the known effects of high fat
diets in breast carcinogenesis.
Toxicology
In long-term toxicology studies, similar problems probably occur. At least one investigator has suggested that diet restriction
be regarded as the norm and more scientific than the currently
used ad lib feeding of rich diets designed for growing young
animals (7).
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animal feed to simulate the 40—60% of dietary calories present
in some high-fat Western diets. However, other nutrients in the
experimental laboratory feeds are usually left at the (high) density levels considered optimum for growth of the young animal.
This may result in the feeding of 'unnatural' levels of nutrients
which could result in laboratory artefacts.
(i) The human RDA (Recommended Daily Allowances) for
nutrients (protein, carbohydrate, energy, vitamins and minerals)
for adults is based on the requirements for optimum health of
the adult (4). In contrast, laboratory animal RDA values are based
on optimum growth of the young animal (9). When dietary intakes of laboratory rodents are compared to humans on the basis
of amount per kg of body wt, some bizarre results can emerge.
In Table I a summary is presented of calcium intake comparisons
between mice, rats and humans. On the basis of mg of calcium
intake per kg body wt there are 30- to 40-fold differences between rodents and man. In the same table are calculations based
on nutrient density, or mg per kcal, which are much more
reasonable and comparable.
On the basis of nutrient density, calcium intake is still several
fold higher (3—8 times) in laboratory rodents then in human diets
in the US. A review of nutrient requirements for domestic farm
animals including poultry, swine, dairy cattle, beef cattle, horses
and dogs, indicates that all have an adult animal requirement for
calcium > 1.5 mg per kcal (10). In addition, diets for a wide
variety of zoo animals also require calcium additions
> 1.5 mg/kcal (11). It would thus seem that calcium intake in
humans in the US is significantly lower, on a nutrient density
basis, than that in laboratory and farm animals.
In a recent study in mice, Bird et al. (12,13) reduced the
calcium level to 0.1 % of the diet, resulting in a nutrient density
of —0.3 mg/kcal, roughly comparable to current human recommended intakes. On oral administration of cholic acid, the irritation and resulting stimulation of epithelial cell proliferation
was much greater on the lower calcium level, as compared with
the 0.5% calcium level normally used in mice. The data suggest
that the higher level of calcium normally used in laboratory rodent feeds may mask a basic characteristic of high fat human
foods, namely the capacity of increased free bile and fatty acids
to irritate the colon and act as promotion agents in colon carcinogenesis. Since diere is a significant segment of the human popula-
Nutrient density
18. Zhang.L., Bruce.W.R. and Bird.R.P. (1987) Influence of dietary fat and
calcium on the proliferation indices of mouse mammary glands. Fed. Proc.,
46, 436.
19. Concensus Conference (1984) Osteoporosis. J. Am. Med. Assoc., 252,
799-802.
20. Research Diets Catalogue (1982) Levels of elements in salt mixtures table.
ICN Nutritional Biochemicals, p. 146.
Received on January 6, 1987; accepted on April 17, 1987
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Summary and recommendations
In recent years, long-term studies in laboratory animals, particularly in tests for carcinogenesis or toxicology, use laboratory
feeds that have been 'standardized' or 'optimized' for maximum
growth of young animals. If continued uniformly through adult
life in long-term studies, such diets can produce abnormal effects such as obesity, and may produce data with little relevance
for application to humans.
It is suggested that diets for animals in laboratory studies in
carcinogenesis and toxicology be adusted to mimic the human
nutrient density of key nutrients (e.g. vitamins and minerals).
This may require a change in diet during the study from a 'rich'
nutrient density required for young growing animals, to the
'leaner' nutrient density of an adult.
The use of nutrient density in designing laboratory animal feeds
to obtain information relative to human conditions is an old
established nutrition concept. Unfortunately, it is often ignored
in current studies, possibly due to the ready commercial availability of diets designed for optimum growth of young animals. A
return to diets that mimic the human in terms of nutrient density
of essential nutrients would produce results more readily useful
and applicable to an understanding of effects in humans.
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