EFFECT GF 5 PERCENT PECTIN N. F. OR 5
PERCENT PECTEN L. M UPON GROWTH.
EXCREYION, SERUM PRQTEMS, AND MENERAL
CGNTENTS EN LEVER AND KIDNEY FESSUES
OF WEANLING MALE RATS OF THE
SPRAGUEQAWLEY HEAEN

Thesis for flu Degree, of pit. D.
fiECHIGkN STATE UNEVERSiTY

Norma Magnus Gilmore
1965

 

THESIS LIBRARY
Michigan Stave
Universit
I ’ I

 

’491. 31133 ~

ABSTRACT

EFFECT OF 5 PERCENT PECTIN N. F. OR 5 PERCENT
PECTIN L. M. UPON GROWTH, EXCRETION, SERUM
PROTEINS,”AND MINERAL CONTENTS IN LIVER
AND KIDNEY TISSUES OF WEANLING MALE
RATS OF THE SPRAGUE-DAWLEY STRAIN

by Norma Magnus Gilmore

Pectic substances are one of the least investigated
and understood classes of carbohydrate in the human dietary.
They are often classified as part of the non-caloric fiber
content of the diet for want of a better place to put them.
Investigators have suggested (1) that ingested pectin
passes through the animal body until it reaches the large
intestine where it is attacked and digested by the flora;
or (2) that as pectic substances are water soluble com-
ponents of plants, they may be digestible carbohydrates and
hydrolyzed and utilized in the animal body. Little quanti-
tative evidence is reported in the literature to support
one or the other of these assumptions.

As it would be of interest to know whether the feeding
of a constant quantity of different pectins could produce
differences in growth and Specific cellular constituents and
functions, pectins similar in molecular weight but signifi-
cantly different in methoxyl content were fed to 150 wean-

ling male rats of the Sprague-Dawley strain. The basal diet

1

Norma Magnus Gilmore

contained 25% casein and in the 2 experimental diets, 5%
citrus pectin N. F. (10 to 12% methoxyl) or 5% citrus
pectin L. M. (5 to 5% methoxyl) replaced 5% of the sucrose.
After 2, 4, and 6 weeks of feeding, animals were randomly
selected from each diet for sacrifice, with the final group
at 8 weeks. Furfural determinations were done as an in-
direct measure of possible absorption of pectins.

The pectin N. F. fed animals consumed less diet and
gained less weight. The pectin N. F. in the diet appeared
to share responsibility for slowing down the rate at which
these animals added weight to the body, and it was necessary
for animals to consume this diet 5 weeks or longer before
this was evident. The feces were the major route of pectin
excretion. There was a sharp difference in the quantity of
uronic acids as galacturonic acid recovered in the excretory
products. Less than 1% of the pectin N. F. consumed could
be recovered while 20 to 25% of the pectin L. M. consumed
was recovered. Although the furfural yielding substances
(FYS) determined in the liver, kidney, and spleen did not
suggest that either pectin was deposited in these tissues,
there was a small but steady trend in the FYS of the liver
that material capable of yielding furfural was increasing
in the livers of the pectin N. F. fed animals and decreasing
in the livers of pectin L. M. fed animals. The FYS per 100

ml of blood paralleled these patterns, respectively.

At 2 weeks, the sera of both pectin fed groups had increased
serum albumin (paper electrophoresis) coupled with a de-
crease in alpha-2 globulin. At 6 weeks, the serum albumin
of pectin N. F. fed animals had decreased, alpha-2 and

gamma globulins had increased, and there was marked retention
of copper (emission spectrograph) by the kidneys. Serum
albumin in rats consuming pectin L. M. tended to decrease
throughout the study and at 8 weeks the alpha-2 globulin was
markedly increased. Magnesium, phosphorus, and zinc in
livers of the pectin L. M. fed rats increased during the
study's last 4 weeks coupled with marked accumulation of
copper in the kidney and later in the liver.

Differences in metabolic response appeared most pro-
nounced for the pectin N. F. fed animals at 6 weeks while
those of the pectin L. M. fed animals seemed to evolve more
slowly and after this point in time. The differences appar-
ently were related to the methoxyl content of the pectins.
There is no strong indication supported by data that pectins
were absorbed and/or hydrolyzed but the metabolic responses
observed cannot be easily explained solely by action of the
microflora upon pectins. More refined techniques need to

be applied to this problem.

EFFECT OF 5 PERCENT PECTIN N. F. OR 5 PERCENT
PECTIN L. M. UPON GROWTH, EXCRETION, SERUM
PROTEINS, AND MINERAL CONTENTS IN LIVER
AND KIDNEY TISSUES OF WEANLING MALE
RATS OF THE SPRAGUE-DAWLEY STRAIN

BY

Norma Magnus Gilmore

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Foods and Nutrition

1965

ACKNOWLEDGEMENTS

The author gratefully acknowledges the assistance and
cooperation given her throughout this study by Dr. Dorothy
Arata, and the graduate students in the Departments of Foods
and Nutrition and Food Science for their help, interest,
critical comment, and encouragement.

A special thanks are extended to Dr. A. L. Kenworthy
of the Department of Horticulture for his help and assistance
in the elemental analyses, and Mrs. Grace Scribner, secretary
of the Department of Foods and Nutrition, for a multiplicity
of activities.

The author knows of no way other than by saying a most
humble thank you to acknowledge Mrs. H. B. Magnus, Mrs.
Elizabeth Smith, Major Jeanne Sherburne, Dr. Esther L. Brown,
Dr. Abigail Hobson, Dr. Seshu Krishna Das, and Dr. Maurice
Richardson for their wonderful and sustaining encouragement
in the face of all difficulties.

And long in the author's memory will remain Dr. B. Elaine
Rutherford, who guided the master's degree, Dr. Beulah Wester-
man, who wished for the author a grand adventure, Dr. Dena
Cederquist, for faith and courage, Dr. Clifford L. Bedford,
who always took time to listen and counsel, and that select few

who remembered red roses.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . . . . . .

Pectic Substances of Plants. . . . . .
Definition and Structure . . . . .
Function . . . . . . . . . . . . .

Plant Pectic Substances in the Body. .
Digestion. . . . . . . . . . . . .
Pectin Sol Infusion Studies. . . .

Animal Studies with Pectic Substances.

EXPERIMENTAL PROCEDURE . . . . . . . . . .

Preliminary Five Week Study. . . . . .
Eight Week Study . . . . . . . . . . .
Methods of Analysis. . . . . . . . . .

RESULTS OF FEEDING STUDIES . . . . . . . .

Preliminary Five Week Study. . . . . .
Results . . . . . . . . . . . . .
Discussion and Summary . . . . . .

Eight Week Study . . . . . . . . . . .
Two Week Metabolism Period . . . .

Results. . . . . . . . . . . .
Summary. . . . . . . . . . . .
Four Week Metabolism Period. . . .
Results. . . . . . . . . . . .
Summary. . . . . . . .“. . . .
Six Week Metabolism Period . . . .
Results. . . . . . . . . . . .
Summary. . . . . . . . . . . .
Eight Week Metabolism Period . . .
Results. . . . . . . . . . . .
Summary. . . . . . . . . . . .

iii

Page

12

12
12
19
23
25
51
41

46

46
47
49

55

55
55
61
65
64
64
68
68
68
72
72
72
8O
80
80
86

TABLE OF CONTENTS - Continued

Page
RESULTS AND DISCUSSION OF EIGHT WEEK FEEDING STUDY . 87
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 112
LITERATURE CITED . . . . . . . . . . . . . . . . . . 119
APPENDIX . . . . . . . . . . . . . . . . . . . . . . 125

iv

TABLE

1.

2.

10.

11.

12.

15.

LIST OF TABLES

Composition of the Basal Diet. . . . . . . . . .

Average Total Food Consumed in Grams by Animals
Fed for 5 Weeks. . . . . . . . . . . . . . . . .

Feces, urine and urinary Furfural Yielding Sub-
stances (FYS) Excreted in 5 Days (TS) by Animals
Fed for 5 Weeks. . . . . . . . . . . . . . . . .

Furfural Yielding Substances (FYS) in Fresh
Liver Tissue from Animals Fed for 5 Weeks. . . .

Furfural Yielding Substances (FYS) and Uronic
Acids as Galacturonic Acid in 5 Day Urine Sam-
ples (TS) and Fecal Excretion of Animals Fed for
2 Weeks. . . . . . . . . . . . . . . . . . . . .

Furfural Yielding Substances (FYS) in Fresh
Liver Tissue from Animals Fed for 2 Weeks. . . .

Protein Components Separated from Sera of
Animals Fed for 2 Weeks. . . . . . . . . . . . .

Furfural Yielding Substances (FYS) and Uronic
Acids as Galacturonic Acid in 5 Day Urine
Samples (TS) from Animals Fed for 4 Weeks. . . .

uronic Acids as Galacturonic Acid in Dried Feces
Excreted in 5 Days (TS) by Animals Fed 4 Weeks .

Furfural Yielding Substances in Fresh Kidney and
Liver Tissue of Animals Fed 4 Weeks. . . .1. . .

Average Total Weight Gained and Food Consumed in
Grams by Animals Fed for 6 Weeks . . . . . . . .

Fresh Kidney, urine Excreted, Furfural Yielding
Substances and Uronic Acids as Galacturonic

Acid in 5 Day Urine Samples of Animals Fed for
6 Weeks. . . . . . . . . . . . . . . . . . . . .

Feces Excreted, Uronic Acids as Galacturonic
Acid in Dried Feces Excreted by Animals Fed for
6 weeks 0 O O O O O O I O O O O O O O O O O O O O

Page

46

55

59

60

65

66

67

7O

7O

71

73

75

76

LIST OF TABLES - Continued

TABLE

14.

15.

16.

17.

18.

19.

20.

21.

ii.

iii.

iv.

Fresh Liver, Furfural Yielding Substances in
Fresh Kidney and Liver Tissues and Blood of
Animals Fed for 6 Weeks. . . . . . . . . . . . .

Protein Components Separated from Sera of
Animals Fed for 6 Weeks. . . . . . . . . . . . .

Emission Spectrograph Data from Liver and Kidney
Tissues of Animals Fed for 6 Weeks . . . . . . .

Urine Excreted, Furfural Yielding Substances and
Uronic Acids as Galacturonic Acid in 5 Day
Samples from Animals Fed for 8 Weeks . . . . . .

Feces Excreted, Uronic Acids as Galacturonic
Acid in Dried Feces Excreted in 5 Days by Animals
Fed for 8 Weeks. . . . . . . . . . . . . . . . .

Furfural Yielding Substances in Fresh Kidney,
Spleen, Liver, and Blood of Animals Fed for
8 Weeks. . . . . . . . . . . . . . . . . . . . .

Protein Components Separated from Sera of
Animals Fed for 8 Weeks. . . . . . . . . . . . .

Recovery of Uronic Acids (GACU) as Galacturonic
Acid in Urine and Feces Excreted in 5 Day

Metabolism Periods by Animals Fed for 4, 6, and
8 Weeks. . . . . . . . . . . . . . . . . . . . .

APPENDIX TABLES

Emission Spectrograph Data for Pectin N. F. and
Pectin L0~ M. O O O O O O 0 O O O O C O O O O O 0

Emission Spectrograph Data of Diets Used in the
Eight Week Study . . . . . . . . . . . . . . . .

Weekly Protein Efficiency Ratios of the Basal
Fed Animals of Each Metabolic Period in the
Eight Week Study . . . . . . . . . . . . . . . .

Weekly Protein Efficiency Ratios of the Pectin
N. F. Fed Animals of Each Metabolic Period in
the Eight Week Study . . . . . . . . . . . . . .

Weekly Protein Efficiency Ratios of the Pectin
L. M. Fed Animals of Each Metabolic Period in
the Eight Week Study . . . . . . . . . . . . . .

vi

Page

77

78

79

82

85

84

85

155

154

155

156

157

LIST OF TABLES - Continued

TABLE

vi.

vii.

viii.

ix.

xi.

Emission Spectrograph Data from
Animals Fed Four Weeks. . . . .

Emission Spectrograph Data from
Animals Fed Six Weeks . . . . .

Emission Spectrograph Data from
Animals Fed Eight Weeks . . . .

Emission Spectrograph Data from
Animals Fed Six Weeks . . . . .

Emission Spectrograph Data from
Animals Fed Eight Weeks . . . .

Milligrams of Nitrogen per Gram
from Animals Fed Eight Weeks. .

vii

Page

Livers of
. . . . . . . . 158

Livers of
. . . . . . . . 159

Livers of
. . . . . . . . 140

Kidneys of
. . . . . . . . 141

Kidneys of
. . . . . . . . 142

of Dried Liver
. . . . . . . . 145

LIST OF FIGURES

FIGURE

1.

2.

5.

Hydrolysis of pectic substances. . . . . . . . .

Weekly weight gained in grams by animals fed for
5 weeks. . . . . . . . . . . . . . . . . . . . .

Average total weight gained in grams by animals
fed for 8 weeks. . . . . . . . . . . . . . . . .

Comparison of average weekly total weight in
grams of animals fed diets containing 5% citrus
pectin N. F. or L. M. for 5 weeks and 8 weeks. .

Milligrams of furfural yielding substances as
percent of basal in 100 mls blood and total
fresh liver tissue from animals fed for 2, 4, 6,
and 8 weeks. . . . . . . . . . . . . . . . . . .

Percent of serum albumin and gamma globulin
separated from sera of animals fed for 2, 4, 6,
and 8 weeks. . . . . . . . . . . . . . . . . . .

Parts per million of copper expressed as percent
of basal in kidney tissue from animals fed for
6 and 8 weeks. . . . . . . . . . . . . . . . . .

Parts per million of copper and zinc, percent of
phosphorus and magnesium expressed as percent of
basal in liver tissues from animals fed pectin

L. M. for 4, 6, and 8 weeks. . . . . . . . . . .

viii

Page
15

57

88

91

98

101

105

106

APPENDIX

METHODS Page

Specifications of Pectin N. F. and Pectin L. M. . . 126
Method of Preparing Diets . . . . . . . . . . . . . 128

Carbazole Method. . . . . . . . . . . . . . . . . . 129

ix

I NTRODUCTI ON

Undoubtedly the diet of the average American will under-
go change in the next fifty years. In light of the expanding
population, Americans by necessity may consume more plant
products in the future. The effect of type, quality, and
quantity of carbohydrates on nutritive status which has been
somewhat ignored by nutrition investigators until recent years
will assume more importance in dietary studies.

Pectic substances are one of the least investigated
and least understood classes of carbohydrates in the human
dietary. This group of high molecular weight structural poly—
saccharides is found in the parenchymatous tissues of fruits
and vegetables such as apples, oranges, turnips, corn, beets,
peas, and beans. Although pectins are recognized constituents
of plant cell walls and tissues and a normal constituent of
food, the contents of the pectic substances are largely un-
known in food materials. Hardinge et a1. ('65) have compiled
the mono, di, and polysaccharide content of a variety of
foods as they are known today and published tables which
should serve as a useful starting point for estimating the
amount of pectic substances ordinarily consumed in the human

diet until knowledge in this area is more complete.

The United States is the largest world producer of
pectin followed by West Germany, Great Britain, Denmark, and
Switzerland. The major source of pectin is fresh peel and
membranes of lemons and oranges followed by apple pomace
and sugar beet pulp. There are two general production
methods for the purification of pectins extracted from these
source materials. One leads to liquid pectin and the other
to a dry pectin. In the latter, pectin is precipitated as
a gel by means of ethanol, calcium carbonate, copper sulfate,
or aluminum chloride and aluminum sulfate. In addition, an
alkali or alkaline salt is added to produce the required pH
for gelation. Thorough washing is necessary to remove
accompanying non-uronide sugars, residues of extraction acid,
salts, fruit oils, acids, and other organic compounds, as
well as metals. The purified pectic substance is then
neutralized with calcium carbonate or ammonia so that a one
percent solution of the product has a pH of 5.5 to 4.0.

This pH appears to provide the greatest stability as long as
the product is kept dry. Bender in 1959 and Kertesz ('51)
have provided excellent summaries of the many production
techniques and testing methods in use by producers and com-
mercial investigators in this field.

Because of outstanding colloidal properties, pectins
have been extensively investigated as by-products. They
have application in such diverse areas as production of

jams and jellies, emulsifying agents for rubber products,

stabilizing agents for ice cream, and coatings for foods,
paper, fibers, and metals. (Kertesz, '51; Stoloff, '58).
Their ability to slowly imbibe water has made them attractive
carrying agents for items which it is desirable to have act
over a period of time. Therefore, they have found applica-
tion as coating materials for pharmaceutical preparations.
Hoffman-LaRoche Inc. ('61) received British patent approval
for a pectin colloid which will contain either an aqueous
solution of vitamins, flavoring agents or growth stimulants.
Pectins are often classified as part of the non-caloric
fiber content of the diet accompanying cellulose (the most
important of the so-called structural polysaccharides),
hemicelluloses, and lignins for want of a better place to
put them. Kertesz ('51) in his review suggests that ingested
pectin passes through the animal body until it reaches the
large intestine where it is attacked and digested by the
microbial flora. But Manville et al. suggested in 1956 that
as pectic substances are water soluble components of plants,
they may be digestible carbohydrates and hydrolyzed and
utilized in the animal body in a manner similar to other poly-
saccharides. Little specific and quantitative evidence exists
in the literature to support one or the other of these
assumptions. However, the literature does describe numerous
“beneficial" therapeutic applications of which anti-diarrheal

and anti-cholesterol are the oldest and the newest.

The apple diet, the oldest therapeutic application of
pectin in this country, was imported from Germany in the
1950s', investigated into the 1940s', and used in the treat-
ment of diarrhea. A variety of reasons were offered for its
effectiveness, and pectin was most often suggested as the
effective agent. Some thought that pectin was bactericidal,
that it could absorb toxins from the intestinal canal, that
released galacturonic acid would be a detoxifying agent, and
that it would be soothing and healing to the inflamed and
ulcerated gastrointestinal tract (Kretscher and Blumberg, '59).

In 1955 Birnberg treated diarrheal conditions in
children with scraped raw apple. Seventy cases were studied
in this way over a year's time in children ranging from nine
months to 12 years of age. The patients exhibited enteritis,
dyspepsia of various origins, or subacute colitis. Of these
cases 88 percent showed marked and rapid improvement in
symptoms in from three to 56 hours. The treatment appeared
to alleviate the symptoms permanently and appeared safe and
practical to use.

Block, Tarnowski, and Green ('59) in studying remedies
for chronic bacillary dysenUary fed six tablespoons of dry
pectin to patients three times a day, and noted that there
was no reduction in temperature and that the course of the
disease was either unaltered or adversely affected. They
concluded that pectin did not serve as a detoxifying agent

in the body. However, when nickel pectinate was administered

to patients suffering from bacillary dysentery at Elgin
State and Dixon State Hospitals, definite improvement was
observed in every patient in appearance and general condi-
tion. Acute symptoms disappeared and patients gained weight.
Attempting to find a more precise means of studying
why the apple diet was effective against diarrhea, Kertesz,
Walker and McCay ('41) formulated a diet which would produce
diarrhea in rats. Weanling animals were used and when fed
10cc of milk containing one gram of lactose every day for a
few days, at least one-half of the animals developed diarrhea.
After the diarrhea persisted for three days, ten grams of
applesauce or another preparation derived from it was added
to the milk-lactose diet. Gross examination of fecal con-
sistency after two days on the diet was used as a yardstick
of cure. Four different supplements were tested for their
effectiveness against the experimentally induced diarrhea.

A. commercial canned applesauce which contained about
0.62 percent pectic substances

B. commercial canned applesauce, pectic substances of
which had been completely hydrolyzed to galac-
turonic acid and methyl alcohol by enzymes

C. pectin extracted from the canned applesauce by hot
water and alcohol, dried, and reconstituted

D. the cellulose—lignin left after the pectin ex—
tractions.

When the various preparations were fed to animals
suffering from experimentally induced diarrhea, 84 percent
of the animals receiving supplement A were cured or greatly

improved, but only 59 percent of those on supplement B.

C was 50 percent effective, and D, 25 percent. The authors
concluded that pectin and not galacturonic acid was the most
effective anti-diarrheal agent in the apple diet.

The latest suggested therapeutic application of pectic
substances is concerned with chronic degenerative disease
rather than acute states. Diet has been among the many
variables investigated in the etiology of atherosclerosis.
Keys and his associates at Minnesota pointed out in 1960
that one of the major differences between the American and
Italian diets was in the complexity of the latter's dietary
carbohydrate. Two diets of natural foods containing 15 per-
cent protein and 16 percent fat in which one had 17 percent
of the carbohydrate calories exchanged in fresh fruits,
vegetables, and legumes, were fed to middle-aged male patients
of Hastings State Hospital. These patients ate first one
diet and then the other for six weeks at a time, and the
diet in which part of the sucrose and milk carbohydrate had
been replaced with fruits, vegetables, and legumes, con-
tained on the average three times more fiber. During the six
weeks in which the men were on the high fiber diet, their
serum cholesterols were lowered, and the difference was
statistically significant. The authors suggested that the
most likely cause of this dietary anti-cholesterol effect
appeared to be the nature of the carbohydrate or some factor

associated with it such as cellulose or pectins.

In order to examine further the effect of these struc-
tural carbohydrates upon serum cholesterol levels in man,
Keys et al. ('61) again fed diets of natural foods to
physically healthy, middle-aged, male patients maintained
under completely controlled conditions in the metabolic unit at
Hastings State Hospital. One series of the study tested the
effect of including 15 grams of cellulose daily in the diet
while another tested the effect of including 15 grams of
citrus pectin N. F. daily. All the diets were similar in
calories, types of fat and cholesterol content. The pectin
or cellulose was incorporated into special biscuits, and
during the control periods, the men ate biscuits similar in
appearance and equivalent in calories. ”When serum cholesterol
data were analyzed, no difference was found in serum choles—
terol level when the men were consuming cellulose supple-
mented diets. However, the serum cholesterol level of these
men was lowered 5 percent when they were consuming pectin
supplemented diets. The anti-cholesterol effect of pectin
was statistically significant at the 0.1 percent level when
the quantity of sucrose consumed was small in relation to the
quantity of legumes and pectin consumed, and was significant
at the two percent level when the ratio was reversed. The
effect of pectin upon the serum cholesterol levels was appar-
ent within three weeks on the diets. It should be remembered
in evaluating this effect of pectin that 15 grams per day

may be at the upper limits of the average dietary consumption

or beyond it. No long term human feeding studies with pectin
have been reported as yet so no statement can be made as to
whether pectin is effective in maintaining lowered serum
cholesterol levels.

Animal studies have also lent support to the apparent
ability of pectic substances in the diet to influence the
levels of serum cholesterol. Wells and Ershoff ('61) main-
tained weanling male rats of the Holtzman strain four to six
weeks on a diet containing 24 percent casein, 61 percent
sucrose, 10 percent cottonseed oil, 5 percent salt mix, and
5 percent citrus pectin N. F. with or without the addition of
1 percent cholesterol. When the pectin N. F. was fed in
conjunction with 1 percent cholesterol, there was a signifi-
cant lowering of the plasma and liver cholesterol and total
liver cholesterol and total liver lipid in contrast to the
levels found in animals not receiving pectin N. F. This
effect was apparent whether the animals were maintained on
the diets four or six weeks or received the supplements each
day or on alternate days. A 5 percent level of pectin in the
diet was found to be more effective than 2.5 percent pectin
N. F. incorporation. The presence of 1 percent sulfasuxidine
and 0.05 percent streptomycin in the rat diet did not alter
the effectiveness of the pectin.

There has been as yet no acceptable explanation why
pectin in the presence of other dietary components exhibits

therapeutic action in either treatment of diarrhea or lowering

of serum cholesterol levels. In 1965 Ershoff suggested

since a 10 percent incorporation of pectin N. F. in the diet
of the rat will prevent experimentally induced atherosclerosis
and Keys et al. ('60; '61) have shown similar cholesterol
lowering effects for man with pectin-containing diets, that
pectin may be an agent for the prevention and treatment of
atherosclerosis. He has suggested that it may be possible to
prepare pectin in suitable concentrates to be used in this
manner.

The suggestion must be viewed with caution and is pre-
mature. Pectin is a variable entity, and its reactions,
because of unique physical qualities and chemical structure,
are influenced by the specific situation in which it is
placed. Relatively little is known about the scope of meta-
bolic effects to be derived from pectin as a dietary con-
stituent. There is uncertainty about the degree to which
pectin may be hydrolyzed in and/or absorbed by the body.
Practically nothing is known about its effect within the
body upon absorption and utilization of other nutrients and
upon cellular activities. In 1942, Hueper studying experi-
mentally induced cardiovascular pathology, listed pectin as
a macromolecular substance capable of producing undesirable
changes within the cells and organs of the body. Possibly,
before there is any unrestrained movement to add pectin
concentrates to the long list of vitamin, mineral, and protein

concentrates available to the consumer, the nutritional

10

aspects of pectin in the animal body should be clarified.

This study was designed to explore metabolic effects
which might be induced by incorporating dry pectin into the
diet of rapidly growing weanling animals. An optimum diet,
similar to that used by Wells and Ershoff, was fed but no
attempt was made to follow the anti-cholesterol effect which
they described. Since it would be of interest to know
whether the feeding of a constant quantity of different
pectic substances could produce differences in growth and
specific cellular constituents and functions, pectins simi-
lar in molecular weight but significantly different in
methoxyl content were fed. The preliminary five week study
was designed to determine whether high and low methoxyl
pectins in the diet of weanling male albino rats would be
excreted in the urine, increase the bulk of the feces, be
deposited in the liver, produce alterations in plasma pro-
tein pattern, or affect excretion of vitamin C which may be
synthesized in rat tissues from galactose and galacturonic
acid. It was followed by an eight week study in which
animals were sacrificed at two, four, six, and eight weeks.

As there are no specific chemical methods available as
yet for isolation of pectic substances in biological materi~
als, the furfural method of Bryant, Palmer, and Joseph ('44)
was used as an indirect assay for possible absorption of

these substances. It was hoped that application of other

11

methods now available, such as paper electrophoresis and

emission Spectrograph, would enable some clarification of

the nutrients and pathways influenced by these substances

in the body.

REVIEW OF LITERATURE
Pectic Substances of Plants

Definition and Structure

Pectic substances were first recognized and investi-
gated by the early French chemists, pharmacologists, and
botanists. Braconnot, a French medical doctor, is generally
credited with first suggesting the therapeutic use of pectic
substances in 1825 (McCready and Owens, '54). Later Swiss,
German, English and then American chemists began investi-
gations into the structure and functions of pectic sub-
stances. Kertesz ('51) in his comprehensive book lists
over 2000 published reports directly concerned with them.

This group of structural polysaccharides is composed
of long chains of predominately d-galacturonic acid units
joined together by alpha 1-4 glycosidic linkages, and is
collectively referred to as pectic substances. In 1944 the
American Chemical Society established a formal nomenclature
for them based upon the recommendations of Baker et al.
Until then terms were often used indiscriminately and inter—
changeably in the older literature so confusion may arise
when reading it today. Protopectin is the name given to the
parent substance which when acted upon by protopectinase gives

rise to pectinic acids. Other members of the pectic

12

15

substances then result from pectinic acids by chain splitting
and splitting-off of branch chains by chemical or enzymatic
means with accompanying production of methyl alcohol.
Eventually, mono, di, and tri-galacturonic acid units and
various non-uronide materials may be obtained. Enzymes
present in plant tissue, fungi, and some bacteria are capable
of splitting the pectic substances into component parts.
Figure 1 with accompanying definitions now in common use may
help clarify the variety of pectic substances.

An extensive literature review concerning the structure
of pectic substances has been done by Joslyn ('62). He sum—
marized the present tentative positions regarding (1) the
structure of the galacturonic acid unit within the chain,

(2) how the side chains may be formed, and (5) how linear
chains may be joined together in the macromolecule.

(1) The hydroxyl groups on carbon atoms C-4 and C-5 of
the galacturonic acid moiety are masked by glyco-
sidic and ring formation; the carboxyl group on
C-6 is either free, esterified with methyl alcohol,
or esterified with araban, galactan, or other
polysaccharides; and carbon atoms 2 and 5 may
be free, esterified with acetyl groups, or linked
by ether-like linkage to polysaccharides or

lignins.

14

 

Definitions |
|
\

Pectic substances: A group designation for those complex,
colloidal carbohydrate derivatives which occur in, ‘
or are prepared from, plants and contain a large
proportion of anhydrogalacturonic acid units which
are thought to exist in a chain-like combination.
Some of the carboxyl groups of polygalacturonic “
acids may be partly esterified by methyl groups'or
partly or completely neutralized by one or more
bases.

 

Protopectin: The water-insoluble parent pectic substance
which occurs in plants and which, upon restricted
hydrolysis, yields pectinic acids.

Pectinic acids: Colloidal polygalacturonic acids contain-
ing more than a negligible proportion of methyl ester
groups. Pectinic acids, under suitable conditions
can form gels (jellies) with sugar and acid or, if
methoxyl content is low enough, with certain metallic
ions. The salts of pectinic acids are either
normal or acid and called pectinates.

Pectin: Water-soluble pectinic acids of varying methyl
ester content and degree of neutralization which
are capable of forming gels with sugar and acid
under suitable conditions.

Pectic acids: Pectic substances mostly composed of col-
loidal polygalacturonic acids and essentially free
from methyl ester groups. The salts of pectic acid
are either normal or acid and called pectates.

15

Figure 1. Hydrolysis of Pectic Substances

Parent: Protopectin

(upon restricted hydrolysis or
enzymatic attack yields)

Pectinic acids

such as

 

Pectin
(High methoxyl pectin
or pectinate)

Higher molecular weight

>= Low ester pectin + methyl alcohol
(Low methoxyl péctin'or pectinate)

 

Lower molecular weight

 

/
Pectic acids (Pectates) + methyl alcohol

Polygalacturonic acids + methyl alcohol

Galacturonic acids +

Methyl alcohol +

Non-uronide sugars: arabinose
galactose
rhamnose
xylose

+
Metallic ions

16.

(2) Side chains may be formed by occasional ester
linkages between carboxyls and free hydroxyls of
polysaccharides; by hemi-acetal linkages between
terminal reducing groups of a polysaccharide chain
and the free hydroxyl of the polygalacturonide;
or by ether linkages between hydroxyls of poly-

saccharide and polygalacturonide.

(5) Linear chains within the macromolecule may be
joined together by hydrogen bonding between two
carboxyl groups; by bonding between non-uronide
sugars; by calcium or other metal ion bonding;
and by bonding of secondary hydroxyl groups by
formaldehyde.

The reader is referred to the review for more extensive
discussion of these structural aspects of the pectic sub-
stances.

As a group then, the pectic substances are hetero-
genous. Protopectin is not water soluble and the other
components are, the molecular weights vary considerably.
there are differences in chain lengths, various suspected
non-uronide materials within the chains, a variety of
attached reactive groups, and differing degrees of methyl-
ation. Kertesz ('51) reports molecular weights of from
20,000 to 280,000. Accurate molecular weight values are
difficult to obtain because the values fluctuate with the

plant tissue used, ease of extraction, amount extracted,

17

chemical and molecular composition of pectic substances in
the tissue, conditions of plant growth, stage of maturity,
storage, enzyme activity, temperature, mechanical injury,
and the method of determination (Joslyn, '62; Joslyn and
Deuel, '65).

In 1949 Jansen and Ward demonstrated that a basic chain
complex contains a minimum of 50 galacturonic acid units.
But there is still question today of whether the molecular
chains are composed strictly of uronic acids or if there are
non-uronide sugars Spotted within the main chains. McCready
and Gee ('60) using thick-paper chromatograph and x—ray
powder diagrams have been able to demonstrate alpha-d-
galactose, beta-l-arabinose, alpha-l-rhamnose, and alpha-
d-xylose in apricot pectin which were apparently incorporated
within the main chain of the polymer. The presence of non-
uronide sugars in the chains would help explain why a gram
of pectinic acid, for example, rarely assays more than 75
to 86 percent galacturonic acid.

Various reactive groups may be attached to the carbons
of galacturonic acid. According to work done by McCready
and McComb ('54), the pectic substances of fruits consist
principally of unbranched chains of galacturonic acid units
which have 80 percent of the carboxyl groups esterified
with methyl alcohol and the remaining 20 percent as free
acids or salts. On the other hand, root vegetables are more

apt to contain pectic substances with free acid groups and

18

polyvalent ions such as calcium, magnesium, or iron. Acetyl
groups may be attached to secondary carbons and McComb and
McCready ('57) have described methods for their characters
ization.

A further measure of pectic substance diversity is in
the degree of methylation. .The ability'of the hydrdgen
of the hydroxyl of C-6 to be replaced by a methyl group
has furnished a convenient criteria by which pectic sub-
stances can be classified and labeled commercially. The
National Formulary defines pectin as a purified carbohydrate
product obtained from the dilute acid extract of the inner
portion of the rind of citrus fruits, or from apple pomace
which consists chiefly of partially methoxylated polygalac—
turonic acid. In order to be classified as a high methoxyl
pectin by their standards, the pectin must contain more than
seven percent methoxyl on an ash and moisture-free basis.
A low methoxyl pectin by definition contains from three to
seven percent methoxyl. If it were possible to completely
esterify polygalacturonic acids, the maximum possible
methoxyl content would be approximately 16 percent with
what is known concerning the molecule itself. But as
Figure 1 suggests, small amounts of non-uronide materials
which may be attached or within the chain affect this value
so that even with the greatest of care in analytical and
extraction procedures, the upper practical limit of methoxyl

content is 11 to 12 percent.

19

Investigators have begun to answer the questions of
the methyl group precursors and the source within the plants
of the carbon skeleton for pectin synthesis. Sato, Byerrum,
and Ball ('57) showed that the tissues of month old radish
plants would synthesize pectinic acid with over 90 percent
of C-14 from administered methionine-C-14 in the methyl
ester on C-6. Using the same plant material, Wu and Byerrum
demonstrated in 1958 that formate-C-14 and glycine 2-C-14
could account for 70 to 80 percent of the C-14 found in the
methyl group on C-6. Seegmiller and co-workers ('56) using
radioactive mono-saccharides observed that the plant may
use either the intact skeleton of glucose or galactose for
synthesis of pectin or a variety of pentoses. So pectic
substances appear to be derived from either galactose or
glucose as the plant tissue undergoes development, and the
methyl groups which give such distinct character to pectic
substances appear to evolve from methyl synthesis and/or

transmethylation.

Function

 

It has been generally held that pectic substances are
the "cementing" material of plant tissues. As such they
are known as the major constituent of the middle lamella,
an amorphous material between primary cell walls. In this
capacity, they supposedly lend additional structure and
support by binding the cellulose walls. Because of their

hydrophilic properties, they could also lend a plastic

20

quality to the cell walls which helps explain the ability
of the cell to expand and elongate. Now that the electron
microscope is available and more specific histochemical
methods can be utilized, it is hoped that the true nature
of the middle lamella and function of pectic substances
can be defined.

Reeve ('59) has reported histochemical and hiStOr
logical observations of immature and ripening Clingstone
and Free-stone peaches in which the cell walls were carefully
measured as the fruit developed. The degree of methylation
of the pectic substances was estimated histochemically using
the reaction of pectin with hydroxamic acids and ferric
chloride developed by McCready and Reeve ('55). The cell
walls of the fruit thickened after mitosis and as the cells
of the fruit enlarged to full size; after which they began
to decrease in thickness as the fruit ripened and softened.
Prior to the end of mitosis, there were no pectic sub-
stances present in the fruit tissues. But as the fruit
enlarged pectic substances were found, and the degree of
methyl esterification remained between 75 and 80 percent
until the green fruit was nearly full size. Just prior to
first stage of ripening, the degree of esterification
approached 100 percent. At the same time that methylation
was maximum, the cell wall thickness was also maximum.
Then as the fruit softened, the cell walls became less thick,
and the degree of methylation among the pectic substances

returned to the earlier level.

21

Using an entirely different approach, Ginsburg ('61)
in some exacting studies on Alaska pea seedlings measured
the degree of cell separation produced by agents such as
EDTA. He has suggested that the middle lamella is a pectin
gel which contains an internal structure of protein mole-
cules cross-linked by two types of metallic ion, the metal-
lic cross-linkage being "chelate in character." He feels
that the stabilization of the middle lamella results from
the protein-metal complex and involves two active sites,
one occupied by monovalent ion, such as potassium, while the
other may contain predominately divalent ions. Calcium,
magnesium, iron, and copper were found to be the best pos-
sible combination for re-cementing separated cells.

Wallace and his co-workers at Purdue ('62), from
studies with Golden Delicious apples, have suggested that
instead of a protein-metal-complex within the gel structure
that the middle lamella may be stabilized by a pectin-metal
or a protein-metal-pectin complex. They determined the con-
tent of amino N, calcium, copper, iron, magnesium, sodium,
and potassium in the water insoluble pulp of fruit picked
at different stages of maturity, and found that magnesium,
iron, and copper as well as calcium were exchanged with
potassium during fruit maturation. At harvest time the de-
crease in polyvalent cations was roughly proportional to the
decrease in protein content. The authors suggested that the

softening associated with ripe fruit may result from

22

dissociation of the protein-pectin-metal complex, whereby
the divalent ions are replaced by hydrogen and potassium.
This would give rise to shorter chains and a less stable

structure.

In process of the Studies with Golden Delicious apples,
Wallace et al. ('62a) inoculated 15 to 20 apples on the trees
with fungi at each harvest period. These fruit were care-
fully watched for the first appearance of rot and this was
recorded as the maturation stage at which the fruit was sus-
ceptible to fungal attack. Interestingly, after the fruit
was susceptible to rot, the insoluble pulp did not contain
the same pattern of metals as pulp from fruit when more
resistant to rot. The quantity of protein, calcium, mag-
nesium, iron, and copper remained higher in the more re-
sistant pulp although the levels decreased as the fruit
matured. The susceptible fruit, however, had a lower protein
level and higher potassium content both of which remained
constant throughout maturation. The activities of the
pectolytic enzymes produced by the fungi were measured using
the dried insoluble pulp of the fruits at each stage of
maturation as the sole carbon source in the mineral media.
The fungi were unable to produce detectable levels of extra-
cellular pectolytic enzymes during growth on the insoluble
pulp from resistant fruit as quickly as on the same pulp from
more susceptible fruit. The authors suggested that the

hypothesized pectin-protein-metal complex in the walls would

25

offer more resistance to hydrolysis by fungal pectolytic
enzymes, and Since the fruit would not be readily available
as a food source, the middle lamella complex could be an
important defense mechanism for immature fruit.

Future investigations may Show that pectic substances
are not inert "cementing" substances but are intimately re-
lated to hydration of the cell walls and tissues and to other
physiological changes associated with growth and which in-

fluence the ripening process of fruit and vegetable tissue.

Plant Pectic Substances in the Body

Digestion

As components of the fruits, vegetables, cereals and
roots of the diet, one takes into the gastrointestinal tract
a cross-section of pectic substances. They are difficult to
isolate and quantify in food materials, and investigators
must eventually evaluate the effect of mixed components as
well as individual components of this group upon cellular
function and on other nutrients and their digestion. But
any investigation into the dietary effects of pectic sub-
stances is faced with the question of whether the higher mole-
cular weight pectic substances are broken down and absorbed
by the body.

E. C. Schneider, under the guidance of L. B. Mendel,

published in 1912 the first nutritional studies of these

24

polysaccharides in the United States. He separated the
water-insoluble components of two kinds of apples and from
this marc prepared pectin by hydrolysis with hydrochloric
acid which yielded 56 percent pentosan and 46 percent
galactan. The mare and the prepared pectin were used by
Schneider in bacterial, enzyme, and human diet studies.

To cultures of peptone, sodium chloride, and two to
three percent marc or one to two percent pectin, Schneider
added one—half to one ml of bacteria suspension prepared by
stirring fresh human feces in sterile water and allowing
the larger particles to settle out. These were cultured
anaerobically or aerobically and allowed to grow for three
to seven days. The cultures showed growth and gas production,
and the bacteria appeared to utilize the pentosan and
galactan fractions of the pectin almost equally well and more
effectively than the marc. The in vitro enzyme studies were
carried out at body temperature with pectin and crude enzyme
preparations including filtered human saliva, diastase, and
alcoholic extract taken from a dog's intestine. No reducing
sugars were found when the mixtures were tested with Fehling's
solution.

In the diet studies, Schneider fed three subjects a diet
free of cellulose for two days, followed by two days on the
same diet supplemented with 40 grams of the raw marc, then
back to two days on the original cellulose free diet. No

other components of the diet were reduced when the marc was

25

added and feces were collected from the subjects for each of
the periods. ‘Since the raw marc was fed rather than the
prepared pectin, Schneider measured the amount of hemi-
celluloses remaining in the feces by assaying for reducing
sugars. Approximately 50 percent of the ingested marc had
disappeared during its passage through the digestive tract.
The urine during this period did not contain reducing sugars
and gave negative tests for pentose. Schneider concluded
from fecal bacterial and enzyme studies, that pectin could
not be broken down by enzymes endogenous to the human digestive
tract and suggested that the apparent hydrolysis observed in
the human feeding studies was due to action of bacterial
flora in the intestinal tract.

Further experimental work was delayed in the digestion
and utilization of these polysaccharides until interest was
stirred in the pros and cons of the apple diet in the treat-
ment of diarrhea. So it was in the 1940's that attempts
were again made to ascertain the metabolic fate of pectic
substances. Kertesz and others used a comparative nutritional
approach in studying this problem with rabbits, mice, dogs,
rats, guinea pigs, as well as human subjects.

For in vitro and in vivo studies with dogs and human
subjects, Kertesz ('40) used a high methoxyl pectin containing
88 percent uronic acid and 10.5 percent methoxyl. During the

in vitro experiments, pH was controlled at 5.4 or 6.4 to

 

avoid,hydrolysis of the pectin by the medium and the temperature

26

at 500C rather than 570C in order to slow down heat denatur-
ation of enzyme protein during prolonged digestion. Saliva
collected from human subjects, dogs, and cows, did not
decompose pectin at either pH during several days of incu-
bation as measured by calcium pectate method. Jejunal
secretions, collected from dogs and very active in sucrase,
amylase, and peptidase, gave no indication of the slightest
digestion of 1.5 percent pectin solutions. Trypsin, pepsin,
rennet, and pancreatic amylase when incubated 12 days did
not decompose pectin.

Kertesz also studied the effect of feces on pectin by
using an aqueous suspension of human or dog feces and incu-
bating it with 1.25 percent pectin solution. At intervals
up to 8 hours, an aliquot taken from the mixture was assayed
for pectin by the calcium pectate method. The majority of
the pectin was rapidly digested by the fecal suSpension
although a small quantity of material would not decompose
after additional prolonged incubation with pectinase. No
other pectolytic enzyme was examined. From his studies,
Kertesz concluded that pectin passes through the digestive
tract without decomposition only to be completely hydrolyzed
by enzymes of the microflora in the large intestine.

Werch and Ivy ('40; '41) studied the fate of ingested
pectin by examining for pectin or its degradation products in
the urine and feces from animal and human subjects on con-
trolled feeding studies. Citrus pectin was fed which yielded

75 percent uronic acid and 9.5 percent methoxyl, and feces

27

were analyzed for uronic acids, furfural, and for pectic
acid by the calcium pectate method.

In the animal studies during the seven day control
period, four dogs were fed a mixed diet consisting of milk,
hamburger, and liver. During the second seven day period,
20 grams of pectin were added daily to the mixed diet.
Pectin alone was fed during the third seven day period.

The feces were pooled from dogs in each period, dried and
then analyzed. The fecal and urine furfural and uronic acid
data obtained when the dogs were in the control period were
subtracted from the values obtained during the experimental
period. Ninety percent of the pectin disappeared during
digestion when it was part of the mixed diet, and during the
third period when pectin alone was fed, only 50 percent dis-
appeared. In this third period, if the animal defecated
frequently only four to 24 percent of the pectin disappeared
during digestion, whereas, if the animal had defecated only
once, 70 to 98 percent of the pectin disappeared. This same
pattern of results was obtained when apple pectin was fed.

In the human studies, six individuals were given a
mixed diet low in crude fiber containing such foods as egg,
white bread, spaghetti, lamb chops, beef steak, potatoes,
and apple pie in the first period. During the experimental
period (all periods were three days), 50 grams of pectin was
added daily to the mixed diet, and in the third ppriOd,,50‘

grams of pectin was taken without food. Since this was a

28

high intake of pectin, the subjects complained of colonic
flatulence, and there was an increase in fecal material.

The data, however, were similar to those obtained with the
dogs. When fed with the mixed diet, 90 percent of the pectin
disappeared during digestion and when fed alone, 85 percent
disappeared compared to 50 percent with the dogs. Werch

and Ivy suggested that pectin decomposition in the intestinal
tract is carried further when pectin is part of a mixed diet,
and that if pectin is retained for a time in the digestive
tract that pectin might have some nutritive value.

Werch and Ivy ('41a) also fed six young men a mixed
diet as previously described but supplemented with 50 grams
of citrus pectin daily. Urine and feces were collected and
one or both were analyzed for volatile acids, total reducing
sugars, and uronic acids. No detectable increase in galac-
turonic acid, reducing sugars, or acetic and formic acids
was found in the urine. There was a tendency toward in-
creased excretion of volatile acids in the feces but they did
not consider this to be significant. No nitrogen determin-
ations were done, but Murer and Crandall had reported in
1940 that normal dogs given doses of 20 to 25 grams of pectic
acid did not excrete increased urinary nitrogen or glucose.

As Werch and Ivy ('42) repeatedly verified the pecto-
lytic activity of fecal suspensions in pectin solutions in
previous studies ('40; '41; '41a), they attempted to isolate

and identify the active bacteria from the materials excreted

29

by animals fed a general diet supplemented with pectin or fed
pectin alone. Interestingly enough, Werch and Ivy noted that
fecal suspensions obtained from dogs on mixed diets actively
decomposed pectin or galacturonic acid but when individual
bacteria isolated from these feces were incubated with pectin
or galacturonic acid, only small quantities of formic and
acetic acids were detected. Although some bacteria required
another carbohydrate source as energy before they could
degrade pectin or galacturonic acid in the culture media,
others required a low oxygen tension. The authors concluded
that the colon was still the most favorable segment of the
gastrointestinal tract for any degradation of pectin. They
attempted to define the nature of the bacterial enzymes sus-
pected responsible for pectin degradation but were unable to
determine whether exo or endo enzymes or both were involved.
One of the curious aspects of the metabolic studies with
pectin was that other investigators were suggesting that
pectin destroyed bacteria rather than the reverse. Haynes
and co-workers ('57) were among those presenting data which
suggested that "pectin" had bactericidal properties. They
prepared a two percent "pectin" solution, added this to Difco
heart infusion broth, adjusted the pH to 5.0 and 5.4, and
after autoclaving inoculated the medium with E. coli. In 48
hours the broth with "pectin" contained no organisms or
the number had decreased at least 98 percent. Various other

‘pHS were tested, and "pectin" showed no inhibitory action

50

above pH 5.5. In 1941 Steinhaus and Georgi verified the
work of Haynes et al. ('57) demonstrating that pectin solu-
tions above pH of 7.0 would not inhibit growth of organisms
of the colon-dysentery-typhoid group. They suggested this
lack of inhibitory ability might be explained by the rapid
decomposition of pectin when it is in weakly alkaline solu-
tion.

A similar but slightly different aspect of this conflict
was reported by Wooldridge and Mast in 1949. They had noted
in some of their clinical work that pectins, pectinates, and
uronic acids when given alone by mouth possessed no potent
in vivo antibacterial properties but became extremely power-
ful bactericidal agents in the gastrointestinal tract when
given together with oral streptomycin. The combination of
pectic substances and streptomycin far surpassed the ability
of streptomycin alone to eliminate intestinal organisms.

They decided to compare the effect of specific pectic sub-
stances alone and in combination with various antibiotics

in vitro. Beta-hemolytic Streptococcus, Staphlococcus aureus,
Pseudomonas aerugenosa, and E. coli were tested in culture
media containing varying concentrations of either seven per-
cent or five percent methoxyl pectins, bismuth, aluminum, or
silver pectinate, or galacturonic acid. All organisms grew
well at pHs of 4.0 or higher. These investigators found

that all compounds tested possessed anti-bacterial activity
and that the cidal effect was not dependent on pH as Haynes

et al. ('57) and Steinhaus et al. ('41) had suggested.

51

Of the agents tested, the most effective were silver pec-
tinate, followed by galacturonic acid, seven percent and
five percent methoxyl pectins. Beta-hemolytic Streptococcus
would not grow in even small concentrations of any of the
pectic substances. When the above substances were set up
with all possible combinations of test organisms except

Beta-hemolytic Streptococcus, and four antibiotics: strepto-

 

mycin, penicillin, fibacitracin, and.tyrothricin, the combi-
nation of each antibiotic with these compounds did produce

a mild degree of synergism but it did not approach that which
had been observed clinically. The authors emphasized that
this synergism between pectin and antibiotics needed a great
deal more evaluation in order to clarify why pectin in pres-
ence of another orally introduced component enhanced the

latter's activity at all.

Pectin sol infusion studies

As World War II was in progress, many macromolecular
substances were suggested as plasma substitutes to increase
blood bulk in the treatment of shock. Studies were under-
taken to determine what happened to large infusions of pectic
solutions in the human body and their degree of safety.

Figueroa and Lavieri reported in 1944 that the infusion
of one percent pectin sols would prevent the onset of experi-
mentally induced shock in dogs for a longer time than would
physiological saline or isotonic glucose solution. There-

were also no after effects observed when one percent pectin

52

sols were administered during appendicitis and gall bladder
operations. Adrenal cortical extract, serum, or pectin sols
sustained blood pressure equally well and a combination of
pectin and adrenal cortical extract was almost as effective
in an animal's resistance to induced shock as serum and
adrenal cortical extract.

Meyer, Kozoll, Popper, and Steigmann ('44) reported
that pectin sols were effective in the treatment of 60 human
patients with shock with no undesirable side effects save for
increased sedimentation rate and pseudoagglutination of some
red blood cells.

These studies on the use of pectin solutions for treat-
ment of shock suggested that the solutions might not be
harmful to the body, but the question of their safety neces-
sitated study at the cellular level. Hueper ('42), investi-
gating the use of macromolecular substances as agents for
development of experimentally induced cardiovascular pathology,
included pectic substances among the molecules tested. From
his extensive studies he developed a "concept of macromole-
cular diseases" with the following "symptom complex": first,
were the evidences of hemodilution which included (1) the
reduction of red blood cells and hemoglobin, (2) increased
sedimentation rate, (5) transitory leukopenia, and (4) per-
sistent leukocytosis or leukopenia; and second, were other
effects, such as (1) impaired coagulation, (2) reduction of

serum proteins, sometimes hyperglobulinemia, and (5) possible

55

development of storage in internal organs depending upon
stability of injected macromolecules. A detailed discussion
of Hueper's ('42) concept was adequately presented by him
elsewhere and only those aspects pertinent to the immediate
study of pectic substances in digestion and the body will be
discussed here.

Hueper ('42a) intravenously injected a one percent
buffered solution of high molecular weight pectins into the
jugular veins of three dogs. One animal received 100 mg
of pectin, another 200 mg of pectin, while the last received
550 mg of the high methoxyl pectin. In the first fifteen
minutes following the injection, there was an increase in
the number of red blood cells per cubic millimeter of blood
followed by a drop to below the original count in four hours.
The red blood cell count than returned to normal levels
within the first 24 hours following injection in animals
receiving 100 or 200 mg of pectin but the red blood cell
count of the animal receiving the 550 mg of pectin continued
a downward trend during the 14 days the animals were under
observation. The sedimentation rate markedly increased after
injection, returned to normal levels within eight hOurs only
to reach high levels again within the next 12 hours and re-
main so for another six to eight days. By the 14th day, the
sedimentation rate was normal again. Prothrombin time re-
mained unchanged, but a moderate shortening of coagulation
time was noted immediately after injection which lasted for

several days. The white blood cells dropped sharply after

54

injection in animals receiving 200 and 550 mg of pectin but
no change was noted in the white cell count of the animal
receiving 100 mg of pectin.

In a second study, three dogs, two of which had been
used in the first study, were given repeated injections of
pectin solution over a 14 week period until the total pectin
received was 25 grams. The animal which had received a 200
mg pectin injection in the first study, showed the same
initial blood picture in this study but while the hemato-
logical pattern returned to within normal limits in the first
two weeks, the sedimentation rate continued at very high
levels throughout the 14 weeks.

In a third study, Hueper studied the effect on tissues
of high molecular weight pectin and depolymerized pectin
solutions. Four dogs and six rabbits received the high
molecular weight pectin solution and another group of three
dogs and Six rabbits received injections of depolymerized
pectin solution. The total pectin received within each group
varied from a minimum of 550 mg to 1580 mg of pectin within
three to 12 weeks. Four of the rabbits who received the 550
mg of pectin died, and when posted, these animals had spleens
four times the normal size and enlarged and mottled livers
and lungs. The remainder of the animals, when sacrificed,
had moderately enlarged spleens and lymph nodes but all other

organs were normal in size, consistency, and color.

55

Tissues were stained with ruthenium-red for histo-
chemical demonstration of pectin. This method indicated
pectin-like material in distended pulmonary capillaries,
foam-cell intima of the aorta, parenchymal and Kupffer cells
of liver, giant cells of the spleen, glomular capillaries
and tubular lumen of the kidney, reticulum cells of the bone
marrow and adrenal. More detailed study of the pulmonary
vessels, renal, myocardial and hepatic arterioles and art~
eries showed irregular distorted nuclei with thickening and
swelling of the lining due to foam-cellular transformation
of endothelial cells. In some instances necrosis of the
foam cells was followed by calcification with or without
thickening. These atheromatous formations apparently result-
ing from the retention of pectin by the endothelial cells of
the arterial intima were less permanent than those previously
observed with cholesterol. The cells appeared to lose their
pectin-like content rapidly and then undergo necrosis or
become fibroblastic. So the primary atheromatous lesions of
which pectin appeared to be a part were rapidly transformed
into secondary sclerotic lesions.

The thyroid, parathyroid and pancreatic tissues were
normal and the stomach and intestines were normal in all
animals except one which possessed several mucosal hemorrhages
in the small intestine. No lesions were found in the cerebral
parenchymal or the vascular system of the brain. Liver,

spleen and aorta tissue Stained for fat with Sudan III were

56

"practically free" of that substance. The hepatic and
splenic cells showed varying degrees of pectin-like storage
and cellular transformations. The changes were more marked
in rabbits than in dogs. In the kidneys, lesions resembled
those seen in certain types of human kidney inflammation.
Swelling and thickening of cortical and tubular tissue and
deposit of pectin-like material accompanied by red blood
cells and other hyaline material was noted in capsular
Spaces.

The tissues from animals receiving the depolymerized
pectin solution Showed almost a complete lack of pectin-like
material in the internal organs and only minor vascular
degenerative changes. The reduction in molecular weight of
the pectin appeared to be of primary importance in the ex-
tent and degree of tissue effect. Hueper noted that the
higher molecular weight pectins have a higher degree of
stability in blood and when absorbed in the cells, may be
somewhat protected for a time against the activities of
cytoplasm. Apparently cells can Slowly remove high molecular
pectin material as long as it has not become too extensive
in the tissues and has not interfered with the normal func-
tioning of the organ. However, he suggested that there is
limited reversibility of macromolecular storage diseases
and other disorders precipitated by macromolecules. If the
deposit persists at an excessive rate or if the arrest of
this process comes too late, the changes in cellular metabol-

ism will be irreversible.

57

In 1945 Popper et al. studied pectin as plasma sub-
stitute according to Hueper's "symptom complex" and in
addition noted that pyrogenic reactions and antigenicity
must be explored also. A total of 155 patients received
injections of from one to nine liters of a one percent
buffered citrus pectin with a molecular weight of approxi-
mately 55,000 (this was thought to be similar to depolymerized
pectin solution used by Hueper in his studies). One-half of
the patients were not in shock and were used as controls,
while the other one-half were in various degrees of surgical,
traumatic, hemorrhagic, and medical shock. Liver biopsies
were obtained from three patients and autopsy material was
obtained from ten patients.

Pronounced evidences of hemodilution were registered
in patients in shock after administration of 1000cc of
pectin solution. When a second liter was administered little
change was recorded and relatively none was noted with a
third liter. So the initial hemodilution effects were not
compounded by additional pectin in the blood. At the same
time the investigators noted that hemodilution effects
attributable to the initial 1000cc of pectin solution con-
tinued long after it was suspected that pectin had been
excreted by the kidneys. The authors attempted to explain
these findings by suggesting that a "certain percentage" of
pectin might remain in the circulation with the first in-

fusion and be maintained by subsequent injections. Or, they

58

suggested that the pectin might start the hemodilution effects
and be replaced by something else which could maintain them
for a longer period of time. As patients with anemia or
hypoproteinemia did not register pronounced hemodilution
effects as did patients with normal blood counts and adequate
protein reserves, they suggested that pectin might initiate
an "autotransfusion" of plasma protein for this mechanism.
Heart, lung, spleen, liver, pancreas and kidney tissue
changes were more pronounced in patients who had received
pectin solutions in excess of the 1000cc ordinarily given for
shock. Splenomegaly and deposition of a peculiar pectin-like
material was observed in phagocytic cells, capillaries,
tissue spaces and around reticulum fibers in the spleen,
kidneys, liver and lungs. Dilatation of glomerular spaces
and tubular lumens of kidneys was noted but the authors sug-
gested that this was a transient Sign and probably insignifi-
cant. Attempts were made to histochemically characterize
the pectin-like deposits in the tissues but no reaction for
amyloid, iron, or calcium was obtained. The pectin solutions
used for infusion in this study resulted in tissue changes
similar to those demonstrated by Hueper in dogs when much
higher molecular weight pectin was injected. The authors
wondered if the pectin-like tissue deposit seen in this study
might not be an admixture of protein, pectin, and other sub-
stances which would have had a higher combined molecular

weight than the pectin injected.

59

In 1944 Bryant, Palmer, and Joseph published a method
designed to determine the quantity of pectic substances
present in biological materials by analyzing for furfural
yielding substances. The method enabled good recoveries
of pectic substances added to biological materials and with
this method, it was possible to follow the transport and
deposition of pectin after large infusions in a more defined
manner.

As a follow-up to their earlier study, Kozoll et al.
('46) determined the furfural yielding substances in blood
and urine of 26 patients who received 15 to 45 grams of
pectin intravenously. Furfural yielding substances in the
blood were determined before the infusion, after adminis-
tration of each 15 grams and at 24 hour intervals thereafter
during the three day observation period. The blood pectin
levels were proportional to the quantity of pectin adminis-
tered being higher in those patients in shock probably due
to their depressed renal function. Hematological changes
previously described somewhat paralleled the level of pectin
in the blood and were maintained much longer than blood
pectin levels. Pectin excretion through the kidneys started
rapidly but decreased after the first day. Those receiving
15 grams of pectin excreted 48 percent of the pectin injected
while those receiving 50 grams, excreted 45 percent by the

end of the three day observation period.

40

Since these studies, Richter ('50) has described
Splenomegaly and focal lesions in liver parenchyma cells,
pectin casts in collecting tubules of kidney, and focal
degenerative changes of various degrees in the renal tubules
of mice injected with high methoxyl pectin and depolymerized
pectin. Small et al. ('50) have described the effect on
rabbit tissue of infusing pectins with molecular weights
varying from 55,000 to 45,000. Animals were killed at inter—
vals varying from one to 122 days after the last injection
of buffered or unbuffered pectin. Lesions, in opposition
to other stadies, caused by treatment with pectin, appeared
to be transitory. They were not seen in tissues beyond the
fourth day after the last injection of buffered pectin and
"slightly longer" with unbuffered pectin. Hartman ('51;

'52) verified Richter's findings using mice who received
lower molecular weight pectin solutions every third day for
21 days. He found that storage of pectin—like material
began promptly and first appeared in the liver, followed by
tubular epithelial of kidney, reticulo-endothelial cells of
lymph nodes, alveoli of lungs, and endothelium of blood
vessels.

As the safety of pectin infusions in the body was
questionable, they were not often recommended by the majority
of investigators for use as plasma substitutes. Just defining
the most beneficial molecule size, degree of methylation, and

best sterilization method would have required much additional

study.

 

41

Animal Studies with Pectin Substances

AS previously described, pectic substances are composed
of galacturonic acid and these have been Shown to play a role
in mucin formation. Kobren et al. ('59) postulated that in-
gested pectin might therefore contribute to maintenance of
mucus membranes chemically as well as physically. In order
to test this possibility, diets containing 18 percent casein
both adequate and deficient in vitamin A and containing
three, six, or 12 percent pectin were fed to female white
'fats to evaluate whether pectin could retard keratinization
of the mucous epithelium and other typical symptoms attributed
to vitamin A deficiency. The animals were examined daily for
Zerophthalmia, and vaginal smears were done to detect the
earliest possible gross cellular changes. When Zerophthalmia
was found, animals were sacrificed and histological studies
were done on eyelids, nares, vagina, liver, esophagus, small
intestine, and kidney. The pectin supplement did not delay
the onset of Zerophthalmia. Marked changes were found in
mucosa and submucosal coats of vagina, nares, and eyelids of
animals on vitamin A deficient diets. However, these changes
were not so marked in animals receiving pectin. The authors
suggested that pectin might be of limited benefit in a
vitamin A deficient diet for maintenance of mucosal integrity.

Several studies were done to ascertain the protective
effect of adding apple to a diet containing lead and/or

arsenic. Among these was a study reported by Murer and

42

Crandall ('42) who used four litter mate pairs of rats to
examine the effect of feeding diets containing 5 percent low
methoxyl pectin upon the excretion of radioactive Pbelo.

The diets contained not more than 0.04 ppm of szlo, and

one member of the pair received 5 percent pectin. Animals
were sacrificed as soon as they consumed 200 grams of diet.
The control animals retained 15.8 percent of the radioactive
lead, excreted 10.9 percent in the urine, and 71.7 percent in
the feces. The pectin-fed animals retained only 11.8 percent
of the lead in their body, excreted less in the urine, 7.9
percent, and more in the feces, 79.8 percent. AS lead will
precipitate pectin in solution, the authors suggested that
the mechanism involved here could be one of inhibiting lead
absorption in the intestine.

While Nath and Meghal ('61) were studying the intestinal
synthesis of thiamine, they fed rats a thiamine-free basal
diet containing 80 percent casein and no carbohydrate and
thiamine-free diets in which 10 percent of the protein was
replaced with 10 percent carbohydrate. Pectin, cellulose,
glucose, sucrose, or honey comprised the carbohydrate content
of the various experimental diets. The weanling rats were fed
for six weeks and the last three days of each week, urine and
feces were collected for thiamine determinations. During the
second, fourth, and sixth weeks, coliform counts were done
on the feces of three rats from each group. Animals were
sacrificed at end of the sixth week and thiamine contents of

liver, kidney, brain, and thigh muscle determined.

43

The data from animals fed the various carbohydrates
were compared with data from the honey-fed group rather than
the non-carbohydrate fed group. The coliform count in the
feces of pectin-fed animals was higher during the first four
weeks. The animals receiving pectin were excreting more
thiamine in the urine and feces after the second week. The
thiamine contents of the liver, brain, kidney, and muscle
tissue were increased at Six weeks, and the differences were
statistically significant. The authors interpreted their
data as indicating that pectin in the diet enhanced the bio-
synthesis of thiamine. They suggested that pectin undergoes
hydrolysis during digestion and that the degradation products
must favorably effect an increase in the intestinal flora.
And they in turn produced more thiamine, some of which was
excreted in the urine and feces. This additional thiamine
was also available to the rats as coprophagy was not prevented.

Had the authors compared the data of the pectin-fed
animals with that from animals on the carbohydrate-free diet,
they would have reported that there was increased thiamine
excretion in the urine only during the third week followed by
decreased urinary excretion of thiamine; there were no dif-
ferences in thiamine excretion in the feces throughout the
Six week period; and that there was significantly less thiamine
present in brain and liver tissue and more present in kidney

tissue.

44

Although there is much in the literature concerning
the pectic substances, there is little concerned with
whether they are digested and absorbed by the body, their
relationship with other nutrients, and effect upon nutritional
status. The many descrepancies in the literature may be
ascribed to feeding periods often too short to allow for
adequate adaptation to experimental diets; some lack of ade-
quate and suitable controls; emphasis upon proteolytic
enzymes or those known to attack fat or starch which could
only be of negligible importance in the breakdown of macro-
molecule of pectin; no clear-cut defining of the bacterial
flora and bacterial enzyme systems suggested responsible
for pectin degradation in the intestinal tract; a histological
picture of undesirable tissue changes based upon a staining
technique, ruthenium red, which has since been replaced by
more specific histochemical techniques for locating pectin in
tissues; and assumptions made without adequately defined data
to back them up.

The disappearance of pectin substances from the gastro—
intestinal tract may be more complex than Schneider's work
indicated. There is some suggestion in the studies of Wool-
dridge and Mast ('49) and Hueper ('42) that the degree of
methylation may be important in the body and cell response to
pectic substances. More recently, Ershoff and Wells ('62)
and Wells and Ershoff ('62) demonstrated that feeding one per-

cent cholesterol in conjunction with 5 percent pectin to the

45

rat was without effect upon plasma and liver cholesterol and
lipids unless the methoxyl content was 10 percent or higher.
Since the rat and not the rabbit, guinea pig, or hamster was
the only animal registering an anti-cholesterol effect, Wells
and Ershoff suggested that the metabolic activities of pectin
may also be species specific. Fausch and Anderson ('65) have
very recently reported that swine fed 5 percent pectin showed
significantly increased concentrations of serum cholesterols,
triglycerides, and phospholipids which are in complete oppo-
sition to studies reported in man and rat.

Any study of pectin substances will be hampered by
inadequate methodology, but as new methods become available
and are applied to this problem, they can shed light on the
questions surrounding the digestion and functional aspects of
pectic substances. The preliminary five week study and the
eight week study reported here made use of some of the newer

methods.

EXPERIMENTAL PROCEDURE

Preliminary five week study

The preliminary study was made during November and
December using 26 weanling male albino rats of the Sprague-
Dawley strain with an average weight of 48 grams. The animals
were randomly distributed to individual wire bottom cages and
housed in animal quarters where the temperature averaged 250C.
Ten animals received the basal diet, eight were fed the basal
diet containing 5 percent citrus pectin N. F., and eight the
basal diet containing 5 percent citrus pectin L. M. The
pectin in the experimental diets replaced 5 percent of the

sucrose. The composition of the basal diet is given in Table 1.

Table 1. Composition of Basal Diet

 

 

Percent (w/W)

 

*

Vitamin-free casein 25
Corn oil ** 10
Mineral Mix 4
Sucrose 61

Vitamin mix, containing the following amounts of
vitamins: 20 mg thiamine-HCl, 80 mg riboflavin,

20 mg pyridoxine-HCl, 60 mg calcium pantothenate,
100 mg niacin, 4 mg biotin, 10 mg folic acid, 400

mg para-aminobenzoic acid, 800 mg inositol, 5 mg
menadione, 2 g chOline chloride, 5000 USP units of
vitamin A, and 5000 USP units of vitamin D was added
to each kilogram of the diet mixture.

 

*
Vitamin-free casein was supplied by General Biochemi-
cals, Chagrin Falls, Ohio.
**

Wesson's salts were obtained from Nutritional Bio-
chemicals Corp. of Cleveland, Ohio.

46

47

The citrus pectins were obtained from Sunkist Growers,
Ontario, California, and the descriptions and specifications
of these two pectins may be found in the appendix, page51126—127.
Diets were made fresh every seven to ten days, and method
of preparation is given in the appendix, pageiUfl3£

The animals consumed diet and ordinary tap water

ad libitum throughout the five week feeding period. They were

 

placed in metabolism cages seven days prior to the end of the
study, and urine and feces were collected. A three day pooled
urine sample from each animal was used for the determination
of furfural yielding substances. Individual urine samples
collected from the animals the last four days on metabolism
were diluted with 0.5M oxalic acid or four percent trichloro-
acetic acid, and frozen for later assay of ascorbic acid by
method of Schwartz and Williams ('55). Animals were sacri-
ficed, and blood and tissues were taken and analyzed as will

be described in the eight week study.

Eight week study

 

In the eight week feeding study, 150 Sprague-Dawley male
albino weanling rats averaging 45 grams were randomly dis-
tributed to individual wire bottomed cages and fed diet and

ordinary tap water ad libitum. They were housed in quarters

 

where the temperature varied between 25°C and 260C during the
eight week study period in the months of July, August, and

September. Fifty animals received the basal diet, another 50

48

received the basal diet containing 5 percent citrus pectin
N. F., while the last group of 50 were fed basal diet con-
taining 5 percent citrus pectin L. M. as described in the

preliminary five week study.

Daily food intake records were kept and weekly weight
gains calculated. At the two, four, six, and eight week
periods, animals were randomly selected from each diet for
study. Thirteen animals were selected from each diet at two
and four week periods, while at the last two periods, 12
animals. During the last three days of the period these
animals were placed in metabolism cages and urine and feces
were collected daily. The urine excreted was collected into
a vessel which contained one ml of toluene. The 24 hour
urine sample of each animal was refrigerated at 4°C until the
total collection period was completed. Then the three samples
of each animal were pooled, frozen, and stored for later
analysis. The feces collected from each animal were allowed
to air dry, then were sealed in glass jars and stored in a
dry, dark area. Animals were weighed the last day of each
period and sacrificed the following day. After anesthetizing
the animals with ether, blood samples of three to 10 mls were
drawn from the abdominal aorta with clean, dry five or 10 ml
glass syringes using #21 or #22 gauge needles. Whenever the
quantity was sufficient, serum and/or potassium oxalated blood
were obtained. Animals were then killed by a blow to the head,

the neck broken, and liver, kidneys, and spleen were excised.

49

The tissues were rinsed in distilled water, blotted dry with
filter paper, and weighed. The spleen and right kidney along
with the small tear-drop caudate lobe situated to the left
beneath the two upper large lobes of the liver were fixed in
10 percent formaldehyde for future histological studies. The
remainder of the liver tissue was homogenized with a Potter-
Elvehjem homogenizer using sufficient deionized water to give
a final volume of 25 to 55 mls. The homogenates were refrig-
erated at 4°C until the end of the sacrifice day, then were
frozen and stored at 0°C. At four and six weeks the left
kidney was homogenized, frozen and stored, and at eight weeks,
the spleen was homogenized, frozen, and stored. A final
volume of 10 ml was used for kidney and spleen tissues.

At the end of the sacrifice day serum was carefully de-
canted from the blood clot, centrifuged at 1000 rpm until
clear (about 20 minutes), stoppered, frozen, and stored.
Protein-free filtrates were prepared from the oxalated blood
samples according to the method of Bryant, Palmer, and Joseph
('44). After the filtrates were centrifuged clear, they were

stoppered, frozen, and stored for later analysis.

Methods of Analysis

The method of Bryant, Palmer, and Joseph ('44) for
determining pectin in biological materials was used with a few
modifications in distillation apparatus and procedure. Urine

samples were thawed and filtered after removal from frozen

50

storage, and pectin was precipitated with 95 percent ethanol.
The tissues homogenized and frozen at time of sacrifice were
removed from storage in a random manner, thawed, and protein-
free filtrates were made from aliquots at room temperature as
described in the method. “

Liver protein-free filtrates were made in triplicate
whereas single filtrates were made from spleen and kidney
homogenates. These protein-free filtrates were then frozen
and stored at 0°C until removed at random for assay. Furfural
yielding substances were determined in the following tissues
and fluids:

at two Weeks: liver and urine

at four weeks: liver, left kidney, whole blood, and urine

at six weeks: liver, left kidney, whole blood, and urine

at eight weeks: liver, left kidney, spleen, whole blood,
and urine.

During the preliminary studies, sufficient samples were
distilled to establish the volume of distillate required to
obtain all furfural yielding substances. For urine and liver
protein-free filtrates all furfural yielding substances were
distilled over in the first 100 mls. Those substances present
in the whole blood distilled over in 25 mls, while 50 and 45
mls were required to obtain all furfural yielding substances
in spleen and kidney tissue, respectively. An additional 10
mls of distillate was collected from each sample and checked

as to completeness of distillation. All tissue protein-free

51

filtrates and urine samples were run in triplicate. Two per-
cent aqueous solutions of each pectin were used as standards.
Furfural yielding substances from pectin N. F. and pectin L.
M. averaged 158 mg per gram.

Maximum absorbancy was obtained at 520 mu using the
Beckman DB spectrophotometer set at narrow slit. Standard
furfural curves were linear between 0.001 and 0.020 mg of
furfural per 10 ml of solution. Aniline for these determin-
ations was purchased in one lot, separated into three dark
brown glass bottles, tightly resealed, and a new bottle was
opened at first indication of deterioration. With each new
aniline, standard curves were re—checked, and the K values
were 0.01756, 0.01772, and 0.01880, respectively, for the
three bottles of aniline.

Urine and feces were analyzed for anhydroglacturonic
acid according to the original method of Dische ('47). The
modified uronic acid carbazole reaction suggested by Bitter
and Muir ('62) was studied but for the particular biological
materials analyzed in this study, the sodium ion seemed to be
more critical than the borate. Therefore, Slight modifications
were made in the reading out procedure of Dische and extraction
procedures were altered to include the sodium ion only. Refer
to appendix for method, page 129.

The uronic acid carbazole determinations were read using
the Beckman DB spectrophotometer, narrow Slit. Maximum

\

absorbancy occurred at 556 mu, and the standard curves were

52

linear between 6 and 40 micrograms per ml of uronic acid.
The carbazole method is sensitive to chloride ions and
necessitates caution in handling of glassware and reagents.
The same lot of concentrated sulfuric acid was used for all
determinations as control on this source of possible impuri-
ties. Standard anhydrogalacturonic acid curves were checked
with each of the new carbazole preparations, and K values
were 98.72, 101.72, and 102.95, respectively. Pectin N. F.,
as fed in the five and eight week studies, assayed by this
method contained 845 micrograms of anhydrogalacturonic acid
per mg or 84 percent, while pectin L. M. contained 705 micro-
grams per milligram or 70 percent. Urine and fecal samples
were run in triplicate wherever possible. uronic acids were
determined in urine collected for all periods, but fecal
uronic acids were not determined on those collected at two
weeks.

The remainder of the homogenized liver and kidney
tissues were dried at 1000C for 11 hours for moisture deter-
minations and elemental analyses. The kidney tissues were
ground by hand in mortar and pestle, and the dried liver
tissue was first ground in a Wiley Mill using a #40 mesh
screen and then reground with a mortar and pestle. All
equipment was thoroughly rinsed in triple distilled and de-
ionized water to eliminate ion contamination.

Five-tenths of a gram of dried liver tissue from animals

sacrificed at the four, six, and eight week periods and

55

0.2 g of dried kidney tissue from animals sacrificed at six
and eight week periods were ashed at 5500F for 12 hours in

a muffle furnace. The ash was diluted 1 ml for every 0.1 g
of dried tissue ashed with 1.8N HCl-Co-Li-K solution and
analyzed for phosphorus,sodium, calcium, magnesium, man-
ganese, iron, copper, zinc, boron, molybdenum, and aluminum
by emission spectrograph.* Five-tenths of a gram of each
diet and each of the citrus pectins were similarly treated
and analyzed for the above elements (appendix, pages 155-154).

Dried liver tissue 'waa assayed in triplicate for total
nitrogen by the boric-acid modification of micro-Kjeldahl-
Gunning method (A.O.A.C., '60).

Blood sera from the animals at all periods were assayed
for protein components by paper electrophoresis using standard
Spinco apparatus and procedure using a current of 10 milliamps
for 16 hours. Strips were stained with bromphenol blue and
scanned in Beckman analytrol.' Slight hemolysis was present
in some serum samples on a random basis but this did not
appear to influence the data obtained. Sera were run in '
triplicate and data for each component were averaged. No sera
were stored more than two months prior to electrophoretic'
separation.

Statistical analysis of the data included standard error
of mean and T-test (Snedecor, '56). In addition, some data

were evaluated by analysis of variance and simple correlation.

A . 1 'Q

*-

1.5 meter "Quantograph" (trade name) photoelectric
spectrometer produced by Applied Research Laboratories, Inc.
Glendale, California.

54

Differences at the 5 percent but not the 2 percent level of
probability were interpreted as indicating a tendency toward
or a trend. Differences at the 2 percent and 1 percent level
of probability were interpreted as indicating significance.
Since throughout the study pectin fed groups were compared
with basal fed groups, Significant differences between pectin_

fed groups will be specifically mentioned.

RESULTS OF FEEDING STUDIES
Preliminary Five Week Study

Results

The protein level of the basal and pectin containing
diets used in the five week feeding study averaged 25.6 per-
cent. The animals accepted the diets well, and there was no
evidence of diarrhea. In the first two weeks of feeding,
the pectin N. F. fed animals tended toward a lowered food
intake. But during the third, fourth, and fifth weeks of the
study no differences in food consumed were noted, and the
total food consumed was not Significantly different. The
food consumption at two weeks and the total food consumed
by the pectin L. M. fed animals was also comparable to that
of the basal and pectin N. F. fed rats (Table 2).

Table 2. Average Total Food Consumed in Grams by Animals
Fed for 5 Weeks

 

_
——

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
*-
At two weeks 152;i 5' 158 i 5 152 i 6
At three weeks 254 i 6 241 i 8 252 i 7
At four weeks 565 i 12 549 i 19 564 i 15
At five weeks 466 i 15 451 i 21 449 i 16

 

Standard error of mean
*
P < .05 > .02

55

56

The basal fed animals averaged a total weight gain of 41 i 1
grams per week during the study period, while the pectin L. M.
fed animals averaged a total weight gain of 40 i 1 grams per
week, and the pectin N. F. fed animals, 57 i 2 grams per week.
The initial average weight of each group was 48 grams but the
pectin N. F. fed animals weighed on the average 17 grams
lighter than the basal fed group at the end of the five week
study. The differences in final weight were not significant.

When the average weight gain per week of each group
was plotted (Figure 2), the pectin L. M. fed animals failed
to gain weight during the second week comparable to the other
four weeks on the study. The pectin N. F. fed animals,
however, having reached an average weekly weight gain similar
to that of the basal and pectin L. M. fed animals at three
weeks, returned to a level similar to their initial average
weekly gain at four weeks, only to return to the former
level at five weeks. No significance could be attached to
these changes in weight gain pattern, however, except to sug-
gest that some adaptation may have taken place at the second
week for animals consuming pectin L. M. and at the third or
fourth week for pectin N. F. fed animals. Any adaptation
may have been concerned with alteration in the microflora
profile introduced by the pectin N. F. and pectin L. M.

Feed efficiency and protein efficiency ratios when ex-
amined on a weekly basis, decreased as would be expected

with rapidly growing weanling rats. The PER of animals fed

57

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58

the basal diet decreased from 2.80 i .05 to 1.55 i .07 at
the fourth week while the PER of pectin N. F. fed animals
decreased from 2.70 i .05 to 1.57 i .09. At the fifth week,
the PERS of pectin N. F. and basal fed animals were 1.65 i
.08 and 1.67 i .09, respectively. The PER of pectin L. M.
fed group, however, decreased from 2.71 r .06 at the first
week to 1.58 i .19 at the second week, maintained this level
through the fourth week, then increased to 1.97 i .11 at the
fifth week. This tendency toward increased weight gain per
gram of protein consumed by the pectin L. M. fed animals
between the fourth and fifth weeks, was noted whether the
data were compared with basal fed or pectin N. F. fed groups.
Whatever the nature of the possible adaptation at the second
week, it did not alter the ability of the pectin L. M.
animals to utilize their diet for growth as efficiently as
other animals.

The fecal and urine excretion of these animals and the
furfural yielding substances contained in the urine are
given in Table 5. The quantities of feces excreted by animals
consuming the two pectin diets were comparable and were
greater than the quantity excreted by basal fed animals.

No observable gross differences were noted in the feces
excreted by the animals on the basal and pectin containing
diets, and no attempt was made to determine the nature of the
increased bulk. Schneider ('12) while studying the effect

of feeding apple marc primarily composed of pectin to three

59

Table 5. Feces, urine and urinary Furfural Yielding Sub-
stances (FYS) Excreted in 5 Days (TS) by Animals
Fed for 5 Weeks

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
***
Urine excreted (ml) 27 i 2.5' 21 i 5.0 17 i 1.0
urinary FYS(mg/TS) 2.05 i .11 2.15 i .19*** 1.91 i .04***
Feces excreted (g) 2.75 i .18 4.52 i .55 4.68 i .57

 

Standard error of mean
*-
P < .01

**
human subjects noted that the apple marc increased the total
amount of feces. Werch and Ivy ('42) suggested from their
feeding studies with citrus pectin N. F. that the pectin
escaping decomposition in the gastrointestinal tract of human
or dog led to increased fecal bulk.

Although the urine excretion appeared to be lower for
pectin N. F. fed animals, the difference was not significant.
The urine excretion of the pectin L. M. fed animals was
significantly depressed after 5 weeks. As no differences
were noted in the weights of fresh kidney taken from the basal
and the pectin fed animals, the depressed excretion may have
resulted from altered function or blockage of tubules by
pectin-like material. No increase in furfural yielding sub-
stances was found in the three day urine samples obtained
from the basal and pectin fed animals. As the latter animals
had free access to pectin-containing diets during the

metabolism period, each group consumed 22.5 grams of pectin

60

during the study, and pectin is water Soluble, it was expected
that if pectin is absorbed to a limited extent that furfural
yielding substances presumably due to pectin would be present
in the urine of these animals. However, as the differences in
fecal quantities excreted by animals on the pectin diets
were statistically significant, the intestinal tract may be
a preferred route for pectin excretion in the rat rather than
urine. ‘

Ascorbic acid was determined in urine samples collected
the last four days of the feeding period. There was a sug-
gestion that pectin fed animals excreted more ascorbic acid
per 24 hours but additional data must be obtained to establish
and verify this excretion pattern. Ascorbic acid levels in
other body tissues during pectin feeding need to be determined.
before any relationship can be suggested.

No significant differences were found among fresh liver
weights and the spleens were not enlarged in pectin fed
animals.

Table 4. Furfural Yielding Substances (FYS) in Fresh Liver
Tissue frbm Animals Fed for 5 Weeks

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Fresh liver (9) 10.59 i .52' 10.75 i .46 10.11 i .45
FYs(mg/g) 1.55 i .09 1.96 i .15* 1.75 i .15
FYS(mg/total fresh 15.95 i1117‘ 20.87 11149**' 17.66 11185‘
liver

 

Standard error of mean
*-

P < .05 > .02
**

P < .02 > .01

61

Per gram of fresh liver (Table 4), the pectin N. F.
fed animals tended toward increased content of furfural
yielding substances at five weeks. When the furfural yield-
ing substances were calculated for the total liver, the
pectin N. F. fed animals showed increased furfural yielding
substances significant at the two percent level. Livers
from animals receiving pectin L. M. contained somewhat higher
values for furfural yielding substances per gram than the
livers of basal fed animals but the difference was not sig-
nificant. The increase in furfural yielding substances
observed in the liver of animals consuming pectin N. F. may
be due to deposit of pectin-like material, but other uronic
acids, pentoses, and a few sugars may have contributed.

Another effect of feeding pectin N. F. and pectin L. M.
was noted in the serum proteins separated by paper electro-
phoresis. A tendency toward decreased serum albumin was
observed in the serum proteins of both pectin fed groups as
compared with those from basal fed animals but no trends or
significant differences were noted in other serum protein

components.

 

Discussion and summary

The preliminary study enabled the refinement of metho-
dology and called attention to the need for additional
information. The growth‘Curves obtained from all three groups

were linear, although pectin N. F. fed animals gained at a

62

lower increment than basal or pectin L. M. fed animals.

There was only a suggestion based upon the changes in aver-
age weekly weight gain pattern that animals consuming pectin
N. F. and pectin L. M. showed some manner of metabolic
adaptation at the second, third, or fourth week. The lack of
furfural yielding substances in the urine and the depressed
urine excretion suggested that the kidney may not excrete
pectin-like material and that kidney function may have been
affected. As the bulk of the feces was Significantly in-
creased, the animals may have excreted pectin in the feces.
The increase in liver furfural yielding substances suggested
that some pectin-like material was being deposited in the
liver. Coupled with this was the tendency toward lowered
percentage of serum albumin as measured by paper electro-
phoresis, which may be indicative of some alteration in liver
cell function.

On the basis of the data obtained in the preliminary
study, a feeding study of longer duration was undertaken to
verify and define the tissue deposit and excretion pattern
in more detail in rats fed 5 percent pectin N. F. or pectin
L. M. AS furfural yielding substances are a relative assay
of pectin and may not detect minor differences in the quantity
of these substances in biological materials, uronic acid
carbazole determinations of urine and feces were included
for comparison and correlative purposes. Although the carba-

zole method is not specific for pectin, it measures uronic

65

acids which are the basic units of pectin N. F. and pectin

L. M. Therefore, the method should be somewhat more indi-
cative of pectin and detect differences in uronic acids in
excretory products of pectin N. F. and pectin L. M. fed
groups if they exist. The method does not measure the uronic
acid of ascorbic acid. In addition to the liver tissue,
kidney and spleen were assayed for furfural yielding sub-
stances. With the assay of urine, feces, and the principle
tissues suggested in the literature as deposit sites for
pectin-like material, it was hoped that some pattern of total
elimination and deposit in relation to pectin consumed could
be established for this group of animals. As the major
structural difference between the pectin N. F. and pectin

L. M. is in the methoxyl content, the chelating ability of
these two compounds might be quite different. Therefore, the
second study included a mineral assay of liver and kidney
tissue. The weanling animals were fed eight weeks to encompass
the most rapid growth phase, and were sacrificed at two week
intervals to more accurately pin-point a possible point of

metabolic adaptation.
Eight Week Study

The diets fed in this study supplied a lower protein
level than those of the five week preliminary study.
Kjeldahl nitrogen determinations of the diets indicated a

protein level of 21.7 percent for the basal and basal containing

64

5 percent pectin N. F. and 22.0 percent for basal containing

5 percent pectin L. M. The basal diet contributed 4.60
calories per gram while the pectin N. F. or pectin L. M. diets
contributed 4.58 or 4.57 calories per gram, respectively.

As animals were sacrificed every two weeks during this
study, the diet groups (basal, pectin N. F., or pectin L. M.)
will be referred to by metabolism period, such as, basal fed
animals of the two week metabolism period. In order to avoid
confusion, the findings of each metabolism period will be

reported separately.

Two week metabolism period

 

Results. Those animals randomly selected from their
respective groups for sacrifice at the close of the two week
metabolism period, Showed no significant differences in total
weight gain or in total diet consumed. Over the two week
period, the basal fed animals averaged a weight gain of 56 i 2
grams per week, the pectin N. F. or pectin L. M. fed animals
averaged 58 i 2 grams per week. The total food consumed by
each group ranged from 115 i 4 grams for the basal, to 120 i
4 grams for pectin N. F. and 125 i 5 grams for the pectin
L. M. groups. No differences were noted in food efficiency
or in protein efficiency ratios.

Furfural yielding substances were determined on the
urine samples collected during the three day metabolism period.

and these and the uronic acid data on the same urine samples

65

are given in Table 5. No significant differences were noted

in the fresh kidney weight or in the quantity of urine excreted
by the animals on the three diets, but differences were noted
in quantity of furfural yielding substances and uronic acids
excreted.

Table 5. Furfural Yielding Substances iFYS) and uronic Acids

as Galacturonic Acid in 5 Day Urine Samples (TS)
and Fecal Excretion of Animals Fed for 2 Weeks

 

 

Basal + 5% Basal + 5%
‘Qiet Basal Pectin N. F. Pectin L. M.
urine excretion (ml) 11.0 i 1.5' 15.0 i 2.4 10.0 i 1.5
FYS (mg/TS) 1.47 1 .10 1.98 i .12*** 1.89 1 .19
Uronic acids (mg/TS) 0.90 i .17 2.84 i .42*** 1.99 i .56**
Feces excreted (g) 0.65 i .05 1.10 i .05* 1.56 i .09***

 

Standard error of mean
*-
P < .05 > .02
**
P < .02 > .01
***
P < .01

The pectin N. F. fed animals excreted significantly more
pectin-like material in the urine whether measured by furfural
yielding substances or uronic acids. Whereas, the furfural
yielding substances in urine of pectin L. M. fed animals was
not significantly different, the uronic acid excretion was
significant at the two percent level of probability. The
quantity of furfural yielding substances or uronic acids ex-
creted by these animals on pectin diets is small in comparison
with the pectin consumed by them during the two weeks of feed—

ing. During the collection of urine and feces these animals

66

consumed on the average 1.5 grams of pectin. The pectin

N. F. fed animals tended to excrete increased quantities

of fecal material, and the pectin L. M. fed group showed
increased excretion of fecal material, statistically Signifi-
cant, when data from these two groups were compared with
those from basal fed animals. No uronic acid determina-
tions were done on this material but on the basis of uronic
acid data from four, six, and eight week metabolism periods,
the increased bulk can be assumed to result from presence

of pectin in the diet.

The feeding of either pectin N. F. or L. M. did not
influence the weight of fresh liver or the milligrams of fur-
fural yielding substances in the liver (Table 6). The lower
furfural yielding substances per gram of liver tissue of
animals maintained upon pectin N. F. or pectin L. M. is of
interest although not significant.

Table 6. Furfural Yielding Substances (FYS) in Fresh Liver
Tissue from Animals Fed for 2 Weeks

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
FYS (mg/g) 4.95 i .59' 4.51 i .45 4.54 i .27
FYS (mg/total fresh 52.61 i 5.4 27.25 i 5.1 28.05 i 2.2
liver)

 

Standard error of mean

67

The animals studied at this metabolism period were
.experiencing rapid growth and liver glycogen stores were
being actively transformed for energy. Pentoses and
occasionally sugars yield furfural substances, and the values
obtained may be reflective of different concentrations of
five and six carbon tissue components and may be indirectly
indicative of a slower mobilization or faster utilization of
energy yielding material in the liver tissue of the pectin
fed animals.

No significant differences were found in the proteins
separated by paper electrophoresis from sera of pectin L. M.
fed animals. But thelsera obtained from pectin N. F. fed
animals Showed a significant increase in serum albumin at
the two percent level of probability, coupled with a tendency
toward decreased alpha-2 globulin (Table 7).

Table 7. Protein Components Separated from Sera of Animals
Fed for 2 Weeks

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Albumin (%) 48.6 4 .6 51.5 4 .9** 51.2 4 1.4
Alpha-1 globulin (%) 14.4 4 .4 15.0 4 .5 14.8 4 .4
Alpha-2 globulin (%) 12.6 4 .5 11.2 4 .5* 11.9 4 .7
Beta globulin (%) 16.1 4 .4 15.5 4 .5 15.6 4 .5
Gamma globulin (%) 8.5 4 .8 7.5 4 .8 6.5 4 .6

 

I
Standard error of mean

*p < .05 > .02
*‘X'
p < .02 > .01

68

The percent gamma globulin appeared to be lower in the pectin
N. F. fed animal sera and still lower in the pectin L. M.

fed animal sera in comparison with the percent found in

the sera of basal fed animals but neither difference was

statistically significant.

Summary. At the two week metabolism period of the eight
week study the pectin N. F. fed animals showed a significant
increased excretion of furfural yielding substances and
uronic acids in the urine, tendency toward increased fecal
excretion, significant increase in percent serum albumin,
and lowered percent serum alpha-2 globulin. Data from the
pectin L. M. fed animals, however, indicated only increased
excretion of uronic acids in the urine and increased excre-
tion of fecal material, significant at two percent and one

percent level, respectively.

Four week metabolism period

Results. Animals maintained for four weeks on the
basal and pectin N. F. or pectin L. M. diets Showed no dif-
ferences in final weight nor in the total quantity of food
consumed. The average weight gain per week for pectin N. F.
animals was 56 i 2 grams the first and second week, then
decreased to 52 i 1 grams by the fourth week. The weight
gain of basal fed animals averaged 56 4 1 grams while the
pectin L. M. fed group averaged 57 i 1 grams for each of the

four weeks. No differences were noted in the quantity of

69

food eaten weekly nor in food efficiency among the three diet
groups. The PER of the basal group decreased from 5.15 i .07
at first week to 1.81 i .11 at the fourth week while the PER
of pectin N. F. fed group decreased from 5.01 i .07 to

1.65 i .08, and PER of pectin L. M. fed group decreased from
5.05 i .09 to 1.72 i .11.

No significant differences were noted in the quantity
of urine excreted by the animals of the three diet groups or
in the weight of fresh kidney. The pectin N. F. fed animals,
however, excreted increased quantities of furfural yielding
substances, significant at the one percent level of prob-
ability (Table 8). The quantity of uronic acids excreted in
the urine of pectin N. F. fed animals was also Significantly
different at the one percent level. Correlation coefficient
of results between these two methods for this group was 0.89.
The pectin L. M. fed animals, although still not showing dif-
ferences in urine furfural yielding substances did show
increased excretion of uronic acids significant at the one
percent level of probability.

Both the pectin N. F. and pectin L. M. fed animals ex-
creted twice the quantity of feces excreted by the basal fed
animals (Table 9). The uronic acids determined per gram of
dried feces tended toward increased significance in the
pectin N. F. fed group but was Significant for the pectin

L. M. fed group.

70

Table 8. Furfural Yielding Substances (FYS) and uronic Acids
as Galacturonic Acid in 5 Day Urine Samples (TS)
From Animals Fed for 4 Weeks

 

Basal } 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
FYS (mg/TS) 1.41 4 .12' 2.15 4 .15*** 1.86 4 .15

Uronic acids (mg/TS) 1.22 4 .16 5.62 4 .52*** 2.59 4 .59***

Correlation coef. 0.50 0.89 0.82

 

Standard error of mean
*
P < .01

**

Table 9. Uronic Acids as Galacturonic Acid in Dried Feces
Excreted in 5 Days by Animals Fed 4 Weeks

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.

H-

.08' 1.62 i .14*** 1.55 1 .Q7***
.29 6.67 i1.39* 188.94 112.0***
.15 8.59 11.51*** 295.91 124.8***

Feces excreted (g) 0.82'

H-

Uronic acids (mg/g) 5.55
Uronic acids (mg/TS) 2.68

|+

 

.Standard error of mean
*-
P < .05 > .02
***
P < .01

Of the 15 pectin N. F. fed animals, there are three not in-
cluded in the averages reported in Table 9. These three
animals excreted 2.55 i .10 grams of feces which contained
51.69, 60.51, and 122.44 milligrams of uronic acid per gram

of dried material. When these values were converted into

71

milligrams of uronic acid per total fecal sample excreted
during the three day metabolism period, values of 75.84,
155.00, and 254.68 milligrams, respectively, were obtained.
These three animals excreted four to 15 times more uronic
acid in feces than the other 10 animals of their group,
and were more comparable to animals of the pectin L. M. fed
group. The latter animals showed significantly increased
excretion of uronic acids per gram of dried feces and in-
creased quantity of uronic acids in the total fecal sample.
Furfural yielding substances determined on the kidney
and liver tissue taken from animals sacrificed at this
metabolism period showed no differences in the quantity of
furfural yielding substances. The data are reported in
Table 10.

Table 10. Furfural Yielding Substances in Fresh Kidney and
Liver Tissues of Animals Fed 4 Weeks

 

ll

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
FYS (mg/g kidney) 0.12 i.005' 0.12 i .005 0.11 4 .006
FYS (mg/g liver) 2.09 4.19 1.88 i .15 1.98 i .14
FYS (mg/total fresh 18.80 41.8 16.56 4 4.5 17.57 41.2
liver)

 

Standard error of mean

No differences were found in the moisture content of liver
'tissue, nitrogen per gram of dried liver, milligrams of
furfural yielding substances in 100 ml of blood, serum pro—

tein components, nor in the quantity of phOSphorus, sodium

72

calcium, magnesium, manganese, iron, molybdenum, copper,
boron, zinc, or aluminum determined by emission spectrograph
in liver tissue. Quantity of calcium, however, was decreased,
significant at the two percent level of probability, when

the values obtained from pectin N. F. fed animals were com-
pared with those of pectin L. M. fed animals. Percent
calcium in livers of pectin N. F. fed animals averaged 0.17 4
.01 while that in livers of pectin L. M. fed animals was

0.20 i .006 and in basal fed animals, 0.19 i .01.

Summary. At the four week metabolism period, both
pectin fed groups were excreting increased quantities of
feces and uronic acids in both urine and feces and the dif-
ferences were statistically significant. The average weekly
weight gain of pectin N. F. fed animals decreased during the
four weeks feeding but was not significant. The only other
difference at this metabolism period was the decreased per-
cent calcium in livers of pectin N. F. fed animals compared

with pectin L. M. fed animals.
Six week metabolism period

Results. Findings of the preliminary five week study
suggested that pectin fed animals may have undergone some
manner of metabolic adaptation prior to sacrifice. In the
eight week study this point of metabolic adaptation or
altered response appeared to center near the sixth week of

the study. The pectin fed animals selected for sacrifice in

75

this metabolism period reacted differently in several ways
from animals sacrificed at two and four week metabolism
periods.

After six weeks of feeding (Table 11), the final weight
of the pectin N. F. fed animals was considerably lower than
the final weight observed in the basal fed and pectin L. M.
fed animals and was statistically significant at the one
percent level of probability. Directly related to this was
a lowered food consumption, also significant at the one per-
cent level.

Table 11. Average Total Weight Gained and Food Consumed in
Grams by Animals Fed for 6 Weeks

*
-

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Weight gained 225 4 11' 174 4 8*** 204 4 8
Food consumed 524 4 19 426 4 17*** 501 4 17

 

Standard error of mean
***
P < .01

When the total weight gain and food consumption data
of the pectin N. F. fed group were broken down into two and
four week feeding periods, the same high degree of signifi-
cance was found for weight gain and food consumed, indicating
that the animals of this metabolism period consumed less food
and gained less weight throughout the feeding period. When
feed efficiency and protein efficiency ratios of the pectin

N. F. fed group were calculated weekly, there was a tendency

74

toward decreased food efficiency ratio and a decreased
protein efficiency ratio significant at two percent at the
second week. However, no differences were noted in these
ratios the other five weeks. Except for the second week,

the randomly selected animals on pectin N. F. diet sacrificed
at the sixth week, apparently utilized the diet as efficiently
for growth as basal and pectin L. M. fed animals throughout
the study. Data comparing the protein efficiency ratios of
the basal, pectin N. F., and pectin L. M. groups at two,
four, six, and eight week metabolism periods, may be found in
the appendix, pages 155-157.

The pectin N. F. fed animals of the six week metabolism
period also tended toward decreased urine excretion coupled
with a decrease in kidney weight. No differences were noted
between groups in the milligrams of furfural yielding sub-
stances in the urine samples, but differences were found in
the uronic acid values (Table 12). The pectin N. F. fed
animals were still excreting an increased quantity of uronic
acid in the urine which was statistically significant, while
the pectin L. M. fed animals were showing increased urinary
uronic acid excretion only tending toward Significance.

The results of the two methods did not show a good
degree of correlation but the uronic acid data were consider-
ed of more importance in measuring the excretion pattern of

pectin-fed animals.

75

Table 12. Fresh Kidney, urine Excreted, Furfural Yielding
Substances and uronic Acids as Galacturonic Acid
in 5 Day Urine Samples of Animals Fed for 6 Weeks

 

m

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Fresh kidney (9) 1.02 4 .04' 0.90 4 .05* 0.97 4 .02
urine excreted (ml) 54 4 6 18 4 5* 27 4 4
FYS (mg/TS) 1.72 4 .19 1.90 4 .11 1.95 4 .11
Uronic acid (mg/TS) 1.16 4 .22 2.65 4 .25*** 1.91 4 .21*
Correlation coef. 0.66 0.66 0.55

 

.Standard error of mean
*

P < .05 > .02

*-

P < .01

**
In contrast to the two previous periods, the pectin N. F. fed
group of the sixth week metabolism period did not excrete in-
creased quantities of feces while the pectin L. M. fed group
maintained the same pattern of excreting increased quantities
of feces (Table 15) as previously observed. Although the
pectin N. F. fed animals were not excreting increased amounts
of feces, they were still excreting increased quantities of
fecal uronic acids as were the pectin L. M. fed animals.
AS in the four week metabolism period, there are two animals
out of the 12 fed the pectin N. F. diet that are not included
in the averages in Table 15. These two animals excreted
1.67 4 .10 grams of feces which contained 55.6 4 .5 milli-
grams of uronic acid per gram. For the total feces excreted

during the three day metabolism period, these two animals

76

excreted seven times more uronic acid than the other members

of the group.

Table 15. Feces Excreted, uronic Acid as Galacturonic Acid
in Dried Feces Excreted by Animals Fed 6 Weeks

 

 

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Feces excreted (g), 1.19 4 .09' 1.40 4 .10 1.96 4.15***
uronic acids (mg/go 2.05 4 .25 6.21 4 .60*** 146.52'46;6***
Uronic acids (mg/ ' 2.51 4 .45 8.01 4 .68*** 295.09 45.1***

total sample)

 

'Standard error of mean.
*-
P < .01

**

No differences were observed in the liver weights, and
the quantity of furfural yielding substances in tissues and
blood presented in Table 14, were not significantly different.
The tendency toward increased furfural yielding substances
per gram of fresh liver in pectin N. F. fed animals noted
in the five week study was not found in the six week metabol-
ism period of this study, although the average furfural yield-
ing substances in the animal tissue from the two studies was
the same, 1.96 4 .15 and 1.95 4 .15. Since pectin N. F.
animals of this particular feeding period did not consume as
much diet and they weighed 16 grams less at six weeks than
the preliminary study group did at five weeks, the average
liver weight was 1.28 grams less. When the total liver
furfural yielding substances are calculated for the pectin

N. F. fed animals of the six week metabolism period, they

77

are lower than those of the pectin N. F. fed animals of the
preliminary five week study. Therefore, the deposit of a
pectin-like material suggested by the findings of the pre-
liminary study is not verified by the data obtained'at the
six week metabolism period of the eight week feeding study.
Table 14. Fresh Liver, Furfural Yielding Substances in

Fresh Kidney and Liver Tissues and Blood of
Animals Fed for 6 Weeks

ll

—__

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Fresh liver (9) 10.89 4.78' 9.47 4 .51 9.87 4 .29
FYS (mg/g kidney) 0.15 4.006 0.15 4 .007 0.14 4 .005
FYS (mg/g liver) 1.71 4.22 1.95 4 .15 1.70 4 .14
FYS (mg/total liver) 20.15 4.5.5 18.45 41.4 16.84 41.5
FYS (mg/100 ml blood) 6.17 4.96 5.55 4 .64 5.41 4 .80

 

Standard error of mean

The percent of serum protein components in the sera
from animals fed pectin N. F. also showed differences although
the serum protein components:in the sera of pectin L. M. fed
animals did not (Table 15). The percent serum albumin of the
pectin N. F. fed animals was decreased significantly greater
than at the one percent level of probability. Coupled with
this lowering of the serum albumin was a tendency toward
increased alpha-2 globulin and an increase in the percent of
gamma globulin. Although a casein diet promotes formation
of body proteins (Allison et al., '59), in general more
efficiently than formation of plasma albumin, the presence

of pectin N. F. in the diet intensified this effect while it

78

apparently stimulated an increase in the alpha-2 globulin
component which represents among others, iron and copper-
protein complexes and lipo-proteins. In addition, presence
of pectin N. F. in the diet of these animals stimulated an
increase in the extra-hepatic component, gamma globulin.
Whether these changes in protein components are indicative
of altered liver cell function and true production of anti-
body or of non-specific gamma globulins needs additional
examination.

Table 15. Protein Components Separated from Sera of Animals
Fed for 6 Weeks

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Albumin (%) 50.2 4 .56' 45.7 4 1.1*** 49.8 4 1.0
Alpha-1 globulin (%) 16.7 4 .4 15.5 4 .6 16.5 4 .6
Alpha-2 globulin (%) 10.2 4 .2 11.4 4 .5 10.7 4 .5
Beta globulin (%) 14.5 4 .2 15.8 4 .4 14.6 4 .5
Gamma globulin (%) 8.5 4 .4 11.7 4 1.0*** -8.4 4 .7

 

'Standard error of mean
-)(-
P < .01

**

Both liver andjkidney tissue from these animals were
analyzed by emission Spectrograph (Table 16). No differences
were found in the quantity of elements in the livers of the
pectin N. F. group but the percent magnesium tended to in-
crease in the livér of the pectin L. M. fed group. The kid—
neys of both pectin fed groups contained increased quantities

of copper, significant at the one percent level, but no other

differences were noted.

79

Table 16. Emission Spectrograph Data from Liver and Kidney
Tissues of Animals Fed for 6 Weeks

 

 

 

 

- Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Liver calcium (%) 0.20 4 .01' 0.17 4 .01 0.19 4 .01
Liver magnesium (%) 0.0714 .002 0.0744 .005 0.0824 .004*
Liver copper (ppm) 25.5 41.4 26.6 41.6 27.5 41.8

Kidney cepper (ppm) 59.4 41.5 61.6 44.9*** 56.4 45.7***

 

Standard error of mean
*-

P < .05 > .02
*-

P < .01

**
As pectic substances are known to chelate calcium, this

was the element which had been expected to Show change within
the tissues if any change could be precipitated by feeding
these two pectins of substantially different methoxyl content.
The pectin N. F. could not be expected to chelate the same
quantity of divalent ion as the pectin L. M. because it con-
tains so many less available hydroxyl groups capable of
losing hydrogen. Therefore, the highly significant increase
in copper rather than calcium in the kidney tissue and by
both pectin fed groups was unexpected. The basal diets con-
tained 15.7 ppm of copper, the pectin N. F. diet 1925 ppm
of copper, and the pectin L. M. diet, 21.5 ppm of copper.
These are not high dietary levels of copper for the rat
(Mills and Murray, '60), and the rat is capable of concen-
trating many times the quantity of copper from a diet con-

taining normal levels of copper without exhibiting any signs

80

of toxicity (Boyden et al., '58). Complete emission
Spectrograph data obtained from livers and kidneys of
animals of each diet group sacrificed at four, six, and eight

weeks will be found in the appendix, pages 158-142.

Summary. The most significant changes noted in the six
week metabolism period, involved the animals consuming the
pectin N. F. diets. These animals consumed less food and
gained less weight, excreted small quantities of pectin-like
material in the urine and the feces but did not appear to be
depositing, as determined by furfural yielding substances,
pectin-like material in the liver or the kidney. The percent
of serum protein components in the sera of these animals sug-
gested a reverse albumin-globulin ratio, and there was
accumulation of copper in the kidney tissue, statistically
highly significant. The animals consuming pectin L. M.
diets were still excreting quantities of feces containing
increased amounts of uronic acids, significant at the one
percent level, and for the first time findings from these
animals indicated increased accumulation of magnesium in the

liver and copper in the kidney, Significant at two percent.
Eight week metabolism period

Results. The pectin N. F. fed animals of the six week
metabolism period gradually consumed less food and gained

less weight, but this was not the pattern found with the

81

pectin N. F. fed animals of the eight week metabolism period.
The pectin N. F. fed animals of this period ceased gaining
weight during the eighth week so that the final weight of
this group (262 4.11) was lower and statistically significant.
No differences were found in the quantity of diet consumed
during the first seven weeks nor the eighth week of the
period. Prior to the eighth week food efficiency and protein
efficiency ratios Showed no differences but at the eighth week
both ratios were significantly decreased. The pectin L. M.
and basal groups consumed the same quantity of diet and
their final weights were 289 i_11 and 298 4.10 grams,
respectively. A tendency toward a decrease in protein
efficiency ratio was noted for pectin L. M. fed animals at
the first and third weeks (appendix, pages 155 and 157),.but no
differences were found among the other weeks of the period.
The decrease in average weekly weight gain observed in the
preliminary study at the second week was not seen in pectin
L. M. fed animals of the eight week metabolism period just
as it was not observed in the two, four, or six week metabol-
ism periods.

The pectin N. F. fed animals no longer excreted in-
creased uronic acids or furfural yielding substances in
the urine (Table 17).

No significant differences were noted in kidney weight
or in the milliliters of urine excreted. The furfural yield-

ing substances and uronic acids in the urine of the pectin

82

Table 17. urine Excreted, Furfural Yielding Substances and
Uronic Acid as Galacturonic Acid in 5 Day urine
Samples from Animals Fed for 8 Weeks

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Urine Excreted (ml) 44 4 9.0' 55 4 8.0 51 4 8.0
FYS (mg/TS) 1.65 4 .16 1.66 4 .14 2.12 4 .14*
uronic acids (mg/TS) 1.51 4 .22 1.95 4 .27 2.69 4 .51*
Correlation coef. 0.85 0.88 0.95

 

Standard error of mean
*
P < .05 > .02

L. M. fed group tended to increase but this may have been
more indicative of the somewhat increased urine excretion
rather than true excretion of pectin-like material. For all
practical purposes, the animals consuming pectin N. F. and
pectin L. M. diets were no longer excreting pectin-like
material in the urine after eight weeks of consuming the
respective pectins in the diet.

The pectin N. F. fed animals did not excrete increased
quantities of feces at the eight week metabolism period but
the pectin L. M. fed animals were still excreting increased
quantities of fecal material (Table 18).

The increased fecal uronic acids as determined in both
pectin fed groups were still statistically significant. In
this period none of the animals in the pectin N. F. fed group
were excreting quantities of uronic acids similar to those

of the pectin L. M. fed group.

85

(Table 18. Feces Excreted, Uronic Acids as Galacturonic Acid
in Dried Feces Excreted in 5 Days by Animals Fed
for 8 Weeks

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Feces excreted (g) 1.40 .09' 1.58 .12 2.52 4 .18***

i
.22 4.50 4 .44*** 102.86 46.8 ***
.29 7.06 41.05*** 258.27 421.9***

Uronic acids (mg/g) 1.89
Uronic acids (mg/TS) 2.59

l+ |+ H-

 

Standard error of mean
***
P < .01

No differences were found in the fresh liver or Spleen
weights or in the quantity of furfural yielding substances in
the liver, kidney, and spleen or blood. However, this was
the first period in which the milligrams of furfural yield-
ing substances per gram of liver tissue of pectin L. M. fed
animals did not compare more favorably with the quantity
found in the livers of pectin N. F. or basal fed animals.

The liver furfural yielding substances of pectin L. M. fed
animals appeared to be inversely related to those values
found in liver tissue of basal or pectin N. F. fed animals.
The high degree of individual variability shown by these
animals throughout the study was especially evident in tissue
assays and has made interpretation of data somewhat difficult

(Table 19).

84

Table 19. Furfural Yielding Substances in Fresh Kidney,
Spleen, Liver, and Blood of Animals Fed for

 

 

 

8 Weeks‘
Basal + 5% Basal + 5%

Diet f Basal Pectin N. F. Pectin L. M.

FYS (mg/g kidney) 0.15 4.008' 0.14 4 .05 0.15 4 .006

FYS (mg/g liver) 1.87 4.16 1.81 4 .26 1.45 4 .20

FYS (mg/total liver)20.98 42.0 20.70 45.8 15.59 42.5

FYS (mg/g spleen) 0.0624.006 0.0654 .008 0.0674 .005
' FYS (mg/100 ml blood)5.24 4.74 4.60 4 .42 4.06 4 .58

 

Standard error of mean

When the sera obtained from both pectin fed groups were
separated into protein components by paper electrophoresis,
the most significant change was noted in the percent of
alpha-2 globulin (Table 20). A tendency toward increased
alpha-2 globulin was noted in the sera of pectin N. F. fed
animals at the six week metabolism period but in the eight
week metabolism period this trend was highly significant.

No differences were noted in any of the other protein com-
ponents. until this particular period there had been no
observable changes in the serum protein pattern shown by the
pectin L. M. fed animals. However, at the eight week metabol-
ism period, the percent of alpha-2 globulin in the sera of
pectin L. M. fed animals was significantly increased at the
two percent level of probability. There was a suggestion

of lowered percent serum albumin but this was not found to

be significant.

85

Table 20. Protein Components Separated from Sera of Animals
Fed for 8 Weeks

 

 

 

Basal + 5% Basal + 5%
Diet Basal Pectin N. F. Pectin L. M.
Albumin (%) 48.0 4 .75' 46.7 4 .92 45.8 4 1.0
Alpha-1 globulin (%) 15.6 4 .5 15.6 4 .6 16.5 4 .6
Alpha-2 globulin (%) 11.5 4 .4 15.4 4 .5*** 12.8 4 .4**
Beta globulin (%) 15.1 4 .5 15.1 4 .2 15.5 4 .5
Gamma globulin (%) 10.0 4 .6 9.5 4 .7 9.8 4 .6

 

Standard error of mean
:**P < .02 > .01
***

P < .01

The most interesting differences among the animals of
the eight week metabolism period were noted in data obtained
by emission spectrograph. The liver tissue of the pectin
L. M. fed animals contained increased quantities of phos-
phorus, sodium, and magnesium significantly greater than one
percent and increased quantities of copper and zinc, signifi-
cant at the two percent level of probability. The liver
tissue of the pectin L. M. fed animals contained increased
quantities of calcium which were significant when compared
with the percent present in liver tissue of pectin N. F. fed
animals. The tabulated data may be found in the appendix,
page140.

. The pectin N. F. and L. M. animals contained an in—
creased quantity of copper in the kidneys at the Six week

metabolism period. A comparable quantity of c0pper was still

86

evident in the kidneys of the pectin N. F. fed groups at the
eight week metabolism period although no longer significantly
different from the basal fed animals. The kidneys of the
pectin L. M. group, however, contained a quantity Similar to
that found in the kidneys of the basal fed group, but the
livers of this group, which were also producing increased
alpha-2 globulin, showed an increase in quantity of copper

at this period, Significant at the two percent level.

Summary. The data obtained during the two, four, and
six week metabolism periods showed most significant changes
occurring in pectin N. F. fed group with a suggestion that
the pectin L. M. fed group might be slower to register change.
At the eight week metabolism period the decreased weight of
the pectin N. F. group was still evident and there was no
excretion of pectin-like material through the urine and no
increase in bulk of feces although the feces still contained
increased quantities of uronic acid. At this period, how-
ever, the pectin L. M. fed animals, who had consistently
excreted quantities of feces containing increased amounts
of uronic acids throughout the study, showed significant
changes in the quantity of magnesium, phosphorus, sodium,
copper, and zinc in the liver and kidney coupled with a

significant increase in the percent of serum alpha-2 globulin.

RESULTS AND DISCUSSION OF EIGHT WEEK
FEEDING STUDY

The five week feeding study suggested that there was a
point in time at which the metabolic responses of weanling
male albino rats consuming diets containing 5 percent pectin
N. F. (high methoxyl) or 5 percent pectin L. M. (low methoxyl)
differed in the parameters tested. In the eight week feeding
study differences in metabolic responses were evident at the
sixth week for pectin N. F. fed animals but those of the
pectin L. M. fed animals seemed to evolve more slowly and
after this point in time. As the pectins in the diets of
these animals, theoretically, differed only in methoxyl con-
tent, the time oriented differences in metabolic responses
observed between these two groups must be related to the
methoxyl content of the pectins.

One of the first indications of difference in response
noted with animals in this study was the final weight achieved
by the pectin fed groups after six or eight weeks of consum-
ing pectin N. F. or L. M. (Figure 5). Arnold ('59) reported
that rats with initial weights of 60 grams showed no differ-
ences in final weights between control and those fed low
methoxyl pectin with 18 percent casein after eight weeks.

The data obtained from pectin L. M. fed animals in both the

87

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89

preliminary five week study and the eight week study reported
here are in agreement with this. However, the pectin N. F.
fed animals of the eight week feeding study did not achieve

a final weight similar to that of the basal fed animals,

just as they did not in the five week study, although the
latter difference was not significant.

Wells and Ershoff ('61) using male rats of the Holtzman
strain gave no indication in their report that animals con-
suming 5 percent citrus pectin N. F. gained less weight dur-
ing the six week feeding study. Those pectin N. F. fed
animals reached an average final weight of 279 grams compared
to 284 grams by the basal fed animals. These weights are
similar to the 262 grams and 298 grams achieved by the pectin
N. F. and basal fed animals, respectively, in this study but
at eight weeks rather than six. It appears that the presence
of 5 percent citrus pectin N. F. in the diet shared responsi-
bility for slowing down the rate at which the animals of the
Sprague—Dawley strain used in this study added weight to the
body, and it was necessary for these animals to consume the
diet five weeks or longer before this was evident.

Zucker ('52) emphasized that two things can lead to
errors in interpretation when one uses the commonly accepted
"rate of gain" in analyzing growth data. First, rats of
different "inherent size“ characteristically have different
growth rates on the same diet. Second, the most rapid rate of

gain is not in a rat with a fully normal history, but in the

90

rat recovering from some setback, such as may occur in the
preweaning period. As the preweaning history is not known
for these animals, little consideration can be given to this
point. Consideration should be given though to the dietary
protein level consumed by the animals. In the five week
study the protein level averaged 25.6 percent while in the
eight week study, diet of similar composition averaged 21.7
percent. Wells and Ershoff ('61) used 24 percent casein but
did not report the protein level of the diet as determined by
Kjeldahl procedure. The rats on the five week study had
initial weights of 48 grams, those of the eight week study,
45 grams, while animals of the Wells and Ershoff study had
initial weights of 45 to 55 grams. Although neither the
dietary protein level nor the initial size as described by
weight alone would be of Significance in the growth rate of
these animals, the animals on the eight week study were
initially somewhat smaller and did consume a diet containing
a slightly lower protein level for a longer period of time.
On the basis of Zucker's comments the animals on the eight
week study could show a rate of growth different (Figure 4)
from those animals of the five week study although they were
of the same strain.

Fisher et al. ('64) reported that two year old cockerels
fed a standard diet containing 5 percent pectin N. F. for 18
months gained one-third as much weight as the control birds.

It would be of interest to know whether a 10 percent casein

Weight in Grams Weight in Grams

Weight in Grams

500‘

200

100‘

45

91 ..—v

Basal
47/

1’
[459’ Figure 4. Comparison of average weekly total
;9’ weight in grams of animals fed diets

48+{7' containing 5% citrus pectin N. F. or

L. M. for 5 weeks and 8 weeks

 

500'

200)

100

48
45‘

r

   
 

Pectin N. F.

 

500 i

200‘

100 4.

48

451

Pectin L. M.

 

 

Hi)
N1)-
(N
.45..
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O)
‘N
CD1:

Weeks

92

diet containing 5 percent citrus pectin N. F., would result
in still greater decreased final weights when fed to weanling
rats, and whether the initial size as described by the weight
of the animals does affect the animal's ability to fully
utilize a diet containing pectin N. F. for growth.

A second indication of difference in metabolic response
exhibited by the pectin N. F. and pectin L. M. fed animals
was in the pattern of eliminating these pectins from the body.
Kertesz, in 1940 stated that "all investigators who have
studied this question agree that pectin added to the diet of
animals and human subjects cannot be recovered from the
feces." In 1941 Werch and Ivy analyzing the feces excreted
by dogs and human subjects on pectin N. F. diets found that
10 percent could be accounted for in the feces when this
pectin was part of a mixed diet.

One of the objectives in the eight week feeding study
was to measure the possible absorption of pectin N. F. or
L. M. eaten by the animals. If it is assumed as Werch and
Ivy ('42) did that pectins in vivo may hydrolyze to uronic
acids as galacturonic acid in the same magnitude as in vitro,
then a relative measure of the degree to which pectin con-
sumed is excreted can be calculated by determining the
quantity of uronic acids as galacturonic acid excreted in the
urine and feces. Since the animals essentially ate the basal
diet altered only with 5 percent citrus pectin N. F. or L. M.,

the uronic acids as galacturonic acid in excretory products

95

of animals maintained on basal diet should be subtracted
from those values obtained from excretory products of animals
maintained on the pectin diets. The resulting differences
should be due to presence of pectin in the diet, and these
data are presented in Table 21.

There was a sharp difference in the quantity of uronic
acids as galacturonic acid recovered in the excretory products
from animals eating these two pectins. Less than one percent
of the pectin N. F. fed could be recovered which is in agree—
ment with Kertesz's statement but not with the findings of
Werch and Ivy. However, differences between species may be
part of the explanation for the lack of agreement as well as
the accuracy with which the methoxyl content of that diet
was defined. As so little of the pectin N. F. was accounted
for in the excretory products, the pectin N. F. fed to these
animals could have hydrolyzed completely in the gastroé
intestinal tract to substances other than uronic acids, been
excreted, and undetected by the methods used; or, the macro-
molecule or its products could have been absorbed, utilized
and/or deposited somewhere in the body. Twenty to 25 per-
cent of the pectin L. M. fed was recovered, so the pectin
L. M. was not fully hydrolyzed in the gastrointestinal tract.
What did "disappear" during the passage of the mixed diet
through the digestive processes may have also been degraded,

absorbed, utilized and/or stored in the body.

94

Table 21. Recovery of Uronic Acids (GACU) as Galacturonic
Acid in Urine and Feces Excreted in 5 Day
Metabolism Periods by Animals Fed for 4, 6, and

 

 

 

 

 

 

8 Weeks
Basal + 5% Basal + 5%
Diet -Basal Pectin N. F. Pectin L. M.
Periods
Four weeks
Diet (a) GACU, mg 1680 1597
urine GACU, mg 1.2 5.6 2.4
Fecal GACU, mg 2.7 8.6 294.0
Total, mg 5.9 12.2 296.4
Total Recovery,% 0.5 21.0
Six weeks
Diet (a) GACU, mg 1421 1260
urine GACU, mg 1.2 2.6 1.9
Fecal GACU, mg 2.5 8.0 295.1
Total, mg 5.7 10.64 295.0
Total Recovery,% 0.5 25.0
Eight weeks
Diet (a) GACU, mg 1278 1289
urine GACU, mg 1.5 1.9 2.7
Fecal GACU, mg 2.6 7.1 258.5
Total GACU, mg 5.9 9.0 241.0
Total Recovery,% 0.4 20.0

 

(a) Pectin N. F. consumed was 2.0, 1.7, and 1.5 grams and
Pectin L. M. consumed was 2.0, 1.8, and 1.8 grams for the
5 day metabolism period of the 4, 6, and 8 week periods,
respectively. The basal diets were not assayed for
uronic acids. One gram of pectin N. F. contained 84%
uronic acid, and one gram of pectin L. M. contained 70%
uronic acid.

95

Trying to correlate the degree of disappearance of these
substances from the intestinal tract with frequency of defe~
cation as Werch and Ivy ('42) reported seemed of somewhat
minor importance and certainly no records of this kind were
kept. Since they found that greater quantities of pectin-like
material were excreted in the feces when pectin N. F. was fed
alone, it would seem more practical to suggest that it took
longer for the pectin N. F. containing diet to take up water
in vivo and to proceed through the digestive processes. If
the mixed diet delayed gastric emptying time, the animals
may have been less hungry and therefore, consumed less diet,
and the pectin N. F. when present in the gastrointestinal
tract may have had increased opportunity to influence other
nutrient absorption, as Fisher et al. ('64) suggested, or to
be hydrolyzed or absorbed, or acted upon by enzymes of the
microflora. Rosenthal and Nasset ('58) reported that fresh
banana used as the carbohydrate source instead of dextrin in
18 percent casein diets produced a slowdown in gastric
emptying time in adult rats. According to Kertesz ('51)
banana contains about 0.59 percent pectic substances on fresh
weight basis, and would have contributed approximately 1.5
percent to the diet used by Rosenthal et al. ('58). The
diets used here contained 5 percent pectic substances.

The pectin L. M. fed animals though, ate quantities of
diet comparable to the quantity consumed by basal fed animals,

and they did consistently excrete more fecal material which

96

contained approximately 21 percent of the pectin consumed.
This picture seems to agree better with increased-defecation
increased pectin-like excretion but may also be related to
solubility of the pectin. Kertesz ('51) states that "a de-
crease in the proportion of esterified carboxyl groups re-
duces the solubility of pectinic acids, although little
quantitative information is available on this point. Pectic
acid, the end product reached upon complete demethylation,
has often been regarded as insoluble in water." The pectin
L. M. fed in this study was esterified with 5 to 5 percent
methoxyl, and had only a few of the unesterified galacturonic
acids chelated calcium, for instance, then portions of the
pectin L. M. could have been insoluble and probably quickly
passed out of the body.

The pattern of uronic acids as galacturonic acid in the
urine and feces of pectin fed groups indicated that the
feces were the major route of pectin excretion for animals
in this study (Table 21). Both pectin fed groups excreted
twice as much fecal material as the basal fed groups during
the first four weeks. The last four weeks of the study, the
pectin N. F. fed groups were no longer excreting increased
amounts of fecal material but the fecal material excreted
contained a similar quantity of uronic acids. The pectin
L. M. fed animals excreted increased quantities of fecal
material throughout the eight weeks which also contained a
relatively constant quantity of uronic acids. Some pectin-

like material was excreted through the urine but for the

97

animals consuming pectin N. F. diets, this mode of eliminat-
ing pectin was of no consequence by the sixth week on the
diet. The pectin L. M. fed animals, excreted a somewhat
constant but small quantity of uronic acids through the
kidneys throughout the study.

AS the nature of the fecal material was not character-
ized beyond its uronic acid content, there is no way of
knowing at the present time what other constituents were in
these feces. Lin et al. ('57) reported increased lipids
and cholesterol in the feces of rats fed cholesterol and
pectin diets as did Fisher et al. ('64) after feeding
standard diets containing pectin to fowl.

Although the furfural yielding substances (FYS) in the
liver, kidney, and Spleen do not suggest that either pectin
was deposited in these tissues, a third indication of dif—
ference in metabolic response between the pectin N. F.
and pectin L. M. fed animals was in the contents of furfural
yielding substances in blood and liver tissue (Figure 5).
For clearer presentation, the quantity of FYS per 100 ml
of blood and per total fresh liver at two, four, six, and
eight weeks were converted to percentage using the FYS
found in blood and liver of basal fed animals as 100 percent.

There was a small but steady indication in the FYS of
the liver that material capable of yielding furfural was
increasing in the livers of pectin N. F. fed animals. It
was not, however, until the eighth Week that the contents of

FYS in the livers of this group reached the level of FYS

98

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99

present in the liver tissue of the basal fed group and
thereby, overcame the initial drop in FYS noted at two
weeks. At the present time, it is not possible to state
whether the suggested increasing content of FYS was due to
deposit of pectin in the liver tissue or due to changes in
other constituents capable of yielding furfural.

Hueper ('42) found histological changes in the liver
tissue suggestive of pectin deposit in both dogs and rabbits
injected with 0.55 grams to 1.4 grams of high methoxyl
pectin over a three to 12 week period. The animals of this
study received pectin orally and might not be expected to
Show histological changes as rapidly. But tissues from
animals of both the five and eight week studies were saved
for histological examination. Possibly when these data
are available, they will clarify the nature of the furfural
yielding material that appears to be increasing in the
livers of the pectin N. F. fed group, and the nature of the
material which was deposited in livers of this group in the
five week study.

The FYS in the liver tissue of pectin L. M. fed animals
showed the reverse trend. The content of these substances
in the liver were also lower than those of the basal fed
group at two weeks; at four weeks the content of FYS in the
liver had increased slightly but for the remainder of the
study, steadily decreased. Therefore, this pectin was not

deposited in the liver tissue of pectin L. M. fed animals.

100

The decreased quantity of FYS in the livers of pectin L. M.
fed animals also raises a question as to the nature of the
carbohydrate in the liver tissue. These animals were gain-
ing weight and growing at a rate comparable to the basal
animals. It may be that other constituents in the liver
capable of yielding furfural were being metabolized quickly
in the livers of these animals in order to maintain this
favorable growth picture and were not available for determin-
ation as pentoses. until longer feeding studies and more
detailed chemical examination of liver tissue is done, there
can only be speculation as to whether the patterns noted in
FYS in the liver tissue from these animals fed pectin N. F.
or L. M. are transitory in nature or indicative of changes
within pentose and uronic acid components of the liver.

An additional indication that the data of FYS in liver
tissues of the pectin fed animals may be of some significance
although not so statistically, is found in the data from
FYS of the blood at four, six, and eight weeks. (These sub-
stances were not determined at two weeks). The FYS per 100
mls of blood parallel the increasing or decreasing pattern
of FYS in the total fresh liver, respectively, in pectin
N. F. and pectin L. M. fed animals.

The changes in serum protein pattern provide a fourth
indication of difference in metabolic response between these
two pectin fed groups (Figure 6; alpha-2 globulins are not
shown). At two weeks, the sera of both pectin fed groups had

increased serum albumin coupled with a decrease in

101

 

 

 

 

 

 

 

 

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102

alpha—2 globulin. The percent of gamma globulin was lower
in sera from pectin N. F. fed animals and still lower in the
sera from pectin L. M. fed animals. At the sixth week the
serum albumin of pectin N. F. animals had decreased and
alpha-2 globulin and gamma globulin had increased. The pro-
tein components in the sera from pectin L. M. fed animals
were undergoing gradual change and presented no outstanding
differences until the eighth week when the only difference
found in sera of either group was an increase in percent of
alpha-2 globulin.

Both hepatic and non-hepatic cells are involved in the
synthesis of the serum proteins. The majority of the serum
proteins are synthesized by the parenchymal cells of the liver
but the reticuloendothelial system, which is concentrated
in cells of the spleen, the lymph nodes, the bone marrow,
and the lungs, synthesize gamma globulin. Weanling rats
apparently do not begin to synthesize gamma globulin until
the twentieth or twenty-first day of life (Engle and Woods,
'60). It may be of some interest that the animals consum-
ing pectin N. F. showed a somewhat lower percent serum gamma
globulin at two weeks and greatly increased percent serum
gamma globulin at Six weeks. The concentration of gamma
globulin in human plasma, for instance, varies more widely
under normal and pathological conditions than that of any
other group of proteins. Janeway and Gitlin ('57) list the
major causes of hypergammaglobulinemia as: antigenic stimu-

lation; infection; hypersensitivity reaction due to the

105

presence of, for instance, foreign material and hepatic
damage- If as Hueper ('52), Popper ('45), Richter ('50),
and Hartman ('51; '52) have suggested, pectin can be stored
in the reticuloendothelial cells of the body and hepatic
parenchymal cells, then the hypergammaglobulinemia noted

at six weeks in this study, could be an indication of such
storage. Or, the hypergammaglobulinemia may be simply a
transitory phenomenon indicating a hypersensitivity reaction
by these animals to a foreign material: pectin N. F.

The increase noted in alpha-2 globulin also needs ad-
ditional clarification and is of further interest because of
its possible relationship to changes which were demonstrated
in the liver and kidney tissue<mfthe pectin N.FH and pectin
L. M. fed animals by emission Spectrograph. Alpha-2 globulin
is a heterogenous component (as is gamma globulin), which
includes glycoproteins, mucoproteins, lipoproteins, thyroxine-
binding proteins, ceruloplasmin, and at least one macro-
globulin. Changes in the percent of alpha~2 globulin may
indirectly indicate that there are changes in the amounts
of these subcomponents. Both iron and copper are complexed
with proteins represented by the alpha-2 globulin of serum
proteins. The iron data obtained by emission Spectrograph
of the liver and kidney tissues were not evaluated since
readings in these tissues were beyond the highest point of
the curve and would have required dilution in order to define
properly. It was thought better to re-evaluate these data

in later studies following liver perfusion techniques. But

104

the copper data along with the elements previously mentioned
were evaluated.

Figure 7, and Figure 8, have been prepared in the same
manner as described for the FYS of the blood and liver tissue.
The parts per million of copper found in the kidney tissue of
the pectin fed animals at six and eight weeks were converted
to percentage using the ppm of copper in the kidney tissue of
the basal fed animals as 100 percent. The kidney tissue of
both pectin fed groups contained increased quantities of
copper at Six weeks. By eight weeks the kidney tissue of
the pectin N. F. fed animals still showed an elevated quantity
of copper but the kidney tissue of the pectin L. M. fed
animals contained ppm of copper similar to that found in the
tissue of basal fed animals. These were the only changes in
the mineral content of kidney tissue noted for either of the
pectin fed groups.

At the same time that the copper content of the kidney
tissue of pectin L. M. fed animals was decreasing, the copper
and zinc contents of the liver tissue of pectin L. M. fed
animals were increasing (Figure 8). Investigators have
pointed out that copper absorption, deposition, and metabol-
ism are influenced by presence of such dietary factors as
zinc, iron, and protein. The values for zinc in the diets
of this study were low, ranging from 17 ppm in the basal diet
to 22 ppm in the pectin N. F. diet. McCall and Davis ('61)

noted that a dietary level of 25 percent protein apparently

105

Figure 7. Parts per million of copper expressed as percent
of basal in kidney tissue from animals fed for
6 and 8 weeks.

Pectin Pectin

N. F. L. M.

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106

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107

eliminated any influence upon the accumulation of copper in
the livers of rats fed even high levels of zinc, 1000 ppm,
and.it is unlikely that the level of dietary zinc in this
study could influence the deposition of copper to any great
extent.

The percent phosphorus and magnesium in the liver
tissue of pectin L. M. fed animals increased during the last
four weeks of the study. No other minerals evaluated in
liver tissue of pectin L. M. fed animals showed changes in
deposition, and the liver tissue of the pectin N. F. fed
animals at four, six, and eight weeks presented no changes
in any of the elements. This last indication of difference
in metabolic response between these two pectin fed groups
may be summarized by pointing out that each of the elements
which accumulated in the tissues of the pectin L. M. fed
animals and not in the tissues of pectin N. F. fed animals,
is essential to the electron transport system and carbo-
hydrate metabolism.

It is difficult on the basis of the findings of this
study to fully explain the changes noted in FYS, serum
proteins, and mineral contents of the liver and kidney
tissue without suggesting that pectic substances in some
form may have been absorbed. It seems possible that metabol-
ism of these substances, exhibiting the heterogenity that
they do, is more complex than can be attributed to the action

of microflora alone upon them. The role of the microflora

108

needs to be defined, however. The urinary uronic acid
values.suggest that at least a small percentage of the pectins
were absorbed and filtered through the kidney during the first
four weeks for the pectin N. F. fed groups and throughout

the study for the pectin L. M. fed groups. However, the
percentage is so small that it is difficult to equate with

the magnitude of the changes.

Methodology was a constant problem in this study. It
may well be that the livers of the pectin fed animals con-
tained more furfural yielding substances than were determined.
The tissues were homogenized at the time the animals were
sacrificed, and although they were sealed and stored at 4°C
until they were frozen, ascorbic acid and hydrogen peroxide
present would have had ready access to any pectins in the
tissues. A better procedure might have been to homogenize
the liver and other tissues just prior to making the protein-
free filtrates. Although the method enables very good
recovery of pectin when it is added to tissues and fluids,
there is no way of knowing just how completely one can
recoVer pectic substances which have undergone the rigors
of digestion and been deposited in tissues. For one thing,
if pectins can form complexes with protein in vivo, they
could be precipitated along with protein during the extraction
procedure. But there is also reason to believe that the
furfural values reported here are dependable. The FYS values

of the blood are most comparable with those reported by

109

Kozell ('46) who used this method. And Bryant, Palmer, and
Joseph ('42), after injecting rabbits for seven weeks with
six percent pectin sols, sacrificed the animals, froze the
liver and kidney tissue whole, homogenized them just prior
to making protein-free filtrates with trichloracetic acid
(instead of sodium tungstate and sulfuric acid as the later
method describes), were still not able to show accumulation
of pectin in liver and kidney tissue by the furfural method.
There is little question that more refined methods will be
necessary in order to define whether pectic substances can
be absorbed from the gastrointestinal tract, and if so, in
what proportion and form, and where they go. Isotope studies
could be of immeasurable value in answering these basic
questions concerning the metabolism of pectins.

The anti-cholesterol effect in man noted by Keys et al.
('61) and in rats by Wells and Ershoff ('61), and the pro-
cholesterol effect in swine reported by Fausche and Anderson
('65) can not be overlooked. Possibly pectin N. F. if it is
not absorbed exhibits not so much a species specific effect
as suggested by Wells and Ershoff ('62a) as its presence in
a mixed diet influences the absorption of monosaccharides
and minerals from the gastrointestinal tract which leads to
a change in the regulation of carbohydrate metabolism.
Although pectin L. M. in the diet has not been shown to have
any effect upon serum cholesterol levels, its enhanced

chelating ability may also be important in altering the

110

absorption of amino acids, minerals, and monosaccharides
from the gastrointestinal tract. And possibly the differ-
ences in metabolic responses evident in this study are the
result of altered absorption.

Hollman ('64) recently reviewed knowledge of the non-
glycolytic pathways of metabolizing glucose and lists concen-
tration of cofactors and inorganic ions and the composition
of the diet as important regulatory factors which may in-
fluence the direction in which glucose breakdown occurs.
Besides glycolysis, two further possibilities for glucose
breakdown in animal metabolism are known. One of these is
the hexosemonophosphate shunt, the other is the glucuronic
acid-xylulose cycle, and the components of both would yield
furfural. If the pentose phosphate cycle does contribute
10 to 50 percent of the glucose breakdown as Hollman states
in his review, then a change in regulation of the three
pathways might reduce, for example, the activity of the
hexosemonophosphate shunt which could result in reduced
generation of NADPH. As NADPH serves as an essential hydro-
gen or electron donor, its decrease or increase would influ-
ence the synthesis of fatty acids and cholesterol and the
resynthesis of hexoses from pyruvic acid in the liver,
among others. It seems of more than passing interest that
the total phosphorus content of the livers from pectin L. M.
fed animals was elevated, and Hollman lists increased concen-
tration of inorganic phosphorus as an inhibitor of the

hexosemonophosphate shunt.

111

For a food component which has been so quickly classi-
fied as part of the~non-nutritive bulk of the diet, a great

deal remains unknown in the metabolism of pectic substances.

SUMMARY AND CONCLUSI ONS

One hundred and fifty weanling male albino rats were
used in this study. They were individually housed in
quarters where the temperature varied between 250 and 260C
during the eight weeks in the months of July, August, and
September when this study was done. Fifty animals received
the basal diet containing 25 percent vitamin-free casein,
10 percent corn oil, 4 percent Wesson salt mix, 61 percent
sucrose, and vitamin mix. The remaining 100 animals randomly
distributed into two groups were fed basal diets containing
5 percent pectin which replaced 5 percent of the sucrose.
One group of 50 animals was fed basal diet containing 5 per-
cent citrus pectin N. F. This is a high quality product
of 10 to 12 percent methoxyl content, commonly referred to
as a high methoxyl pectin. The third group of 50 animals
received the basal diet containing 5 percent citrus pectin
L. M. This is also a product relatively free of impurities
containing only three to five percent methoxyl, and can be
called a low methoxyl pectin. The animals consuming one or
the other of the pectin diets will be referred to as pectin
L. M. fed rather than low methoxyl and pectin N. F. fed
rather than high methoxyl.

Daily food intake records were kept and weekly weight

gains recorded. After two, four, and six weeks of feeding,

112

115

animals were randomly selected from each diet for sacrifice,
with the final group sacrificed at eight weeks. At the end
of the first two periods, a total of 59 animals were sacri-
ficed, while at the last two periods, 56 animals. They were
placed in metabolism cages for three days prior to sacrifice
and urine and feces were collected. Blood samples were
drawn from the abdominal aorta. Then the livers, kidneys,
and spleen were excised. The furfural method of Bryant,
Palmer, and Joseph was used to identify possible pectin-like
material in biological tissues and fluids. The feces were
analyzed for uronic acids by modification of the carbazole
method of Dische. Serum proteins were separated by paper
electrophoresis Spinco procedure, and strips were scanned
by Beckman analytrol. Dried liver and kidney tissues were
analyzed by emission Spectrograph for phosphorus, sodium,
magnesium, manganese, copper, zinc, iron, boron, aluminum,
calcium, and molybdenum. The data were statistically evalu-
ated by T-test and analysis of variance.

The initial weight of each of the groups was 45 grams.
The final weight of the basal group averaged 298 grams, the
pectin N. F. group averaged 262 grams, and the pectin L. M.
fed group, averaged 289 grams. The basal and pectin L. M.
fed groups consumed Similar quantities of the respective
diets. The pectin N. F. fed animals, however, gained less
weight and the difference was statistically significant at the

one percent level of probability. There was an indication

114

by the fourth week of the study that pectin N. F. fed animals
were gaining less weight. ‘This pattern was pronounced at the
Sixth week, and the pectin N. F. fed animals failed to gain
weight during the eighth week. The pectin N. F. fed animals
consumed less diet but when food efficiency and protein
efficiency ratios were calculated weekly, the only difference
found was in the eighth week. Although consuming less food
and gaining less weight, the pectin N. F. fed animals appeared
to utilize the diet for growth with the same relative
efficiency as the pectin L. M. and basal fed animals.

During the first four weeks of feeding, no differences
were found in the quantity of urine excreted, and both pectin
L. M. and N. F. fed groups were excreting significantly in-
creased quantities of uronic acids in the urine. However,
after six and eight weeks of consuming the respective pectins,
these animals no longer excreted pectin-like material in the
urine. During the first four weeks both pectin fed groups
were also excreting twice as much fecal material as the basal
fed groups, and these feces contained a significantly in-
creased quantity of uronic acids per gram. During the last
four weeks, the pectin N. F. fed group were still excreting
increased quantities of uronic acids in the feces, but the
amount of fecal material was similar to that of the basal fed
animals. The pectin L. M. fed group eliminated increased
quantities of feces high in uronic acids throughout the eight

weeks. In this study, the feces rather than the urine

115

appeared to be the primary route of pectin excretion,
especially for the pectin L. M. fed group.

There was a sharp difference in the quantity of uronic
acids as galacturonic acid recovered in the excretory products
of these animals with regard to quantity of pectin N. F. or
L. M. consumed by them. Less than one percent of the pectin
N. F. fed to these animals could be recovered while 20 to 25
percent of the pectin L. M. fed was recovered. As no dif—
ferences were found in the furfural yielding substances in
the blood, liver, Spleen, or kidney tissue of the pectin fed
animals at the two week intervals of the study, the body
apparently did not deposit these pectins.

At two weeks, the sera of pectin fed animals showed a
significant increase in percentage of serum albumin coupled
with tendency towards a decrease in the percentage of alpha-2
globulin. The percentage of gamma globulin was lower in the
sera of pectin N. F. fed animals and still lower in the sera
of pectin L. M. fed animals but these were not statistically
significant. At the sixth week the percentage of serum
albumin of the pectin N. F. fed animals had decreased signifi-
cantly greater than at the one percent level of probability
and coupled with this was a tendency toward increased per-
centage of alpha~2 globulin and a significant increase in the
percentage of gamma globulin. By the eighth week, the only
difference in the serum proteins was an increase in alpha-2

globulin in sera of both pectin fed groups.

116

The major difference between the pectins fed to these
animals was in the methoxyl content and possible chelating
ability. Theoretically, there should be more replaceable
hydrogen in the low methoxyl or pectin L. M. The divalent
ion most commonly associated with pectins is calcium, and
this was the ion expected to Show change within the tissues
if.any change in tissue content could be precipitated by feed-
ing these two pectins. The data obtained were not signifi-
cant for calcium. The kidney tissue of both pectin fed
groups contained significantly increased quantities of copper
at six weeks. By eight weeks the kidney tissue of the pectin
N. F. fed animals still showed an elevated quantity of copper
although it was no longer significant, and the kidney tissue
of the pectin L. M. fed animals contained ppm of copper
Similar to that found in the tissues of basal fed animals.
But the liver tissue of the pectin L. M. fed animals was now
showing at the eighth week a Significant increase in copper
content.

The ppm of zinc found in the liver tissue of the pectin
L. M. fed animals showed no differences during the four and
six weeks of feeding but did show an increase at eight weeks
significant at two percent level of probability. The percent
phosphorus found in the liver tissue of the pectin L. M. fed
animals gradually increased during the last four weeks of
“the study as did the percent of magnesium, and by eight weeks,

the increased content of both was statistically Significant.

117

No differences were found for any of the elements assayed
in the liver tissue of the pectin N. F. fed animals after
four, six, or eight weeks of feeding.

The weanling male albino rats consuming diets contain-
ing 5 percent pectin N. F. or high methoxyl, or 5 percent
pectin L. M. or low methoxyl showed differences in metabolic
response in the parameters tested centered near the sixth
week of the eight week feeding study. While these differences
in metabolic response appeared to center at this point for
the pectin N. F. fed animals, those of the pectin L. M. fed
animals seemed to evolve more Slowly and after this point in
time. The differences in metabolic response were apparently
related to the methoxyl content of the pectins.

A diet containing pectin N. F. or L. M. did not appear
to be harmful to the rat but the metabolic responses observed
in this study cannot be easily explained solely by action of
the microflora upon pectins. Whether or not pectins taken
orally, can be absorbed from the gastrointestinal tract and
by the cells of the rapidly growing body, is crucial to a
full understanding of the different metabolic responses ob-
served between the two pectin fed groups. Important, also,
is whether pectic substances in a mixed diet can influence
the absorption of amino acids,monosaccharides, and minerals
from the gastrointestinal tract, possibly through biological
chelation (Rubin and Princiotto, '65), which might lead to

a suggested change in the regulation of carbohydrate

118

metabolism, and/or the differences in metabolic response

observed in this study. Considering the heterogeneity of

pectic substances, they may exhibit more than one mode of

action. More refined techniques need to be utilized to

define the metabolic fate of pectic substances.

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t”4\h_._¥

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Werch, S. C., and A. C. Ivy 1940 On the fate of ingested
pectin. Proc. Soc. Exp. Biol. Med., 44: 566.

 

1941 On the fate of ingested pectin. Am. J. Dig.
Dis., 8: 101.‘ ' ‘ ' '

1941a A study of the metabolism of ingested pectin.
Am. J. Dis. Child., 62: 499.

 

, R. W. Jung, A. A. Day, T. E. Friedemann and A. C.
Ivy 1942 The decomposition of pectin and galacturonic

acid by intestinal bacteria. J. Infectious Dis.,
70: 251.

 

Wooldridge, W. E., and G. W. Mast 1949 Effects of uronic
acids, pectins and pectinates on the enteric flora,
alone and in combination with antibiotics. I.
In vitro studies. Am. J. Surg., 78: 881.

Wu, P. H. L., and R. U. Byerrum 1958 Biosynthesis of
pectinic acid methyl esters. Plant Physiol., 55: 250.

Zucker, L. M. 1952 National Vitamin Foundation. Rat
Quality: a consideration of heredity, diet, and
disease; proceedings of the symposium held at Columbia
Univ. College of Physicians and Surgeons. New York,
Jan. 51, p. 12.

AP PENDI X

125

 

126

SPECIFICATIONS OF PECTIN N. F. AND PECTIN L. M.

For the citrus pectins obtained for this study, Sunkist

Growers supplied the following descriptions and specifications.
Sunkiet Brand Pectin N. F., High Viscosity (product No. 5442)

This is a high viscosity product that may be of value
to pharmaceutical manufacturers. It meets all National
Formulary Specifications and is a yellowish-white, practically
odorless product. It is almost completely soluble in twenty
parts of water at 25°C, forming a viscous, opalescent,
colloidal solution which flows readily and is acid to litmus.
It is insoluble in alcohol or in dilute alcohol, and in other
organic solvents. There is not more than:

10% loss on drying (1050C for 2 hours)

4% ash

0.4% acid-insoluble ash
5.5 ppm arsenic

5 V ppm lead

Not less than: 7% methoxyl content on a moisture and
ash free basis, approximately 9 to 11.5%

78% galacturonic acid content on a moisture and ash
free basis.

Pectin N. F. No. 5442 contains not more than 20 milli-
grams of sugars and organic acids per 125 milligrams of

pectin, and it passes National Formulary test for starch.

127

The product is especially adapted to liquid prepara-
tions where a high viscosity product is indicated and may
possibly reduce or eliminate non-therapeutic viscosity
agents such as gums.

The Sunkist Growers did not supply a similar listing
for the Pectin L. M. but the Specifications should be some-
what the Same. The major difference between the two pectins
is in methoxyl content, and the pectin L. M. contains a much
lower percentage. The supplier did send the following

description.

Exchange Brand Pectin L. M. (a low methoxyl pectin, Product
No. 5466)

This pectin is described as a pectin with a low ester
content which can be standardized for its calcium gel form-
ing ability. It will form gels with milk or fruit juices
and in the dietetic field, is incorporated with foods and
beverages, particularly in diets where sugar is replaced by
non-nutritive sweeteners. Approximate methoxyl content is

5.5 to 5%.

 

' - *AOA'J—

128

METHOD OF PREPARING DIETS

Preliminary work with the diets indicated that pectins
tended to form hard balls unless the diet was mixed in a
Specific manner. The following procedure for incorporating
the various components of the diets was found to give the
greatest uniform quality and diet was totally acceptable to
the animals. They were unable to distinguish pectins from

the balance of the diet and ignore them.

., ‘ i .Miillim

Corn oil and fat soluble vitamins were allowed to mix
in bowl of an 8 kilogram Hobart mixer. Sucrose was added,
mixed in well, then aqueous solution of choline chloride,
water soluble vitamins, and approximately 50 cc of distilled
water were added. When these were well mixed, Wesson's
salts and casein followed. After these were adequately in-
corporated, the appropriate pectin supplement was slowly
added while the mixing continued. All diets were refriger-
ated in glass or metal containers. When metal containers l
were used, they were lined with cellophane or plastic bags E
so diets were kept as free from contact with metal as !

possible.

129
CARBAZOLE METHOD
Extraction of urinary Uronic Acids

The pectin—like material present in two, three or four
mls of filtered undiluted urine*was precipitated with 95
percent ethanol as described in the furfural method of
Bryant, Palmer and Joseph ('44). The precipitation and the
subsequent extraction of urinary uronic acids were carried
out in 50 ml centrifuge tubes and in triplicate wherever
there was sufficient quantity of urine. The urine-ethanol
mixture stood at room temperature for at least two hours,
and during this period, the tubes were vigorously Shaken by
hand three times to aid the clumping of flocculant material.
After centrifuging the tubes at 1000 rpm for 50 minutes, the
supernatant was decanted and discarded. Five mls of 60 per-
cent ethanol were then mixed with the precipitate, and after
standing 15 minutes at room temperature, the tubes were
recentrifuged at 1000 rpm for 15 minutes. This procedure
was repeated, the supernatant being decanted and discarded
each time. After the second ethanol wash, the tubes were
turned upside down on filter paper to drain.

The precipitates were redissolved in three mls of triple
distilled water, shaken vigorously, and allowed to stand for
15 minutes before centrifuging at 1000 rpm for 15 minutes.
The supernatant was decanted into a 10 ml volumetric flask.

This procedure was repeated twice, once with four ml of 0.05

150

N NaOH and finally with two mls of triple distilled water,
and then the flask was made up to 10 mls with triple dis-
tilled water. A small amount of precipitate which could not
be solubilized by the procedure was negative for galacturonic
acid when tested with basic lead acetate. The 10 mls of
extracted uronic acids were stoppered and stored in the

refrigerator overnight and read in the manner to be described.

Extraction of Fecal Uronic Acidsi

The feces, which were air dried following collection
during the three day metabolism periods, were finely ground
in a Wiley mill using a #60 mesh screen, redried in a 1000C
oven for two hours, and stored in a dessicator until weighed.
Weights of the fecal samples used for uronic acid extractions
from animals on pectin containing diets varied between 100
and 150 mgs and from animals on basal diets between 160 and
200 mgs., and samples were set-up in triplicate._ The feces
from basal fed animals were so low in uronic acids, that it
was necessary to determine the fecal uronic acids by the
addition of one mg of pectin L. M. or pectin N. F. to each
sample. The uronic acids contained in the pectin were then
subtracted from the total uronic acid values found in the
feces containing the added pectin. Triplicates checked
within five percent.

The weighed feces (in 50 ml centrifuge tubes) were

moistened with three ml of 60 percent ethanol. Ten ml of

151

triple distilled water was well mixed with the feces and
after standing 50 minutes, the Sample was centrifuged for
15 minutes at 1000 rpm. The supernatant was decanted into
a 100 ml standardized graduated cylinder. This procedure
was repeated twice using 10 mls of triple distilled water
each time. Samples were then treated in the same manner
with 10 or 20 mls of 0.05N NaOH until a final volume of 70 i

to 150 mls was extracted. The fecal uronic acids from

 

basal fed animals were collected in 50 mls. A final 10 mls )
was collected in the same manner from each sample, stored

separately, and analyzed for uronic acids to be sure that

all uronic acids had been collected in the 50 or 70 to 150

ml extractions. A 20 ml aliquot from each collection was

decolorized with 500 mg of a 1.5:1 mixture of charcoal and

celite, and refiltered. The latter was used for carbazole

determination of uronic acids by the method of Dische ('47).

Modification of Dische Procedure L

 

The procedure as described by Dische was essentially i

A;

followed. The sulfuric acid was stored at 40C throughout
the running of the samples. The tubes were packed in ice
during the addition of all reagents and were returned to an
ice bath following the 20 minute boiling period. Carbazole
was not added until the samples were at room temperature.
The samples were stored in the dark for two hours and then

read using the Beckman DB spectrophotometer set a narrow slit

152

with maximum absorbancy obtained at 556 mu. Spot check of
these samples-with 5.8cc of distilled water as well as by
color verified the presence of uronic acids rather than
hexoses.. As much as 60 gammas of glucose were not found

to interfere in the range of anhydrogalacturonic acid

standards used.

 

 

155

Table 1. Emission Spectrograph Data for Pectin N. F. and
Pectin L. M.

 

 

 

Elements Pectin N. F. Pectin L. M.
%~Phosphorus 0.027 0.025
ppm Sodium 5120 2568
% Calcium 0.15 0.10
% Magnesium 0.00 0.00
ppm Manganese 8 8
ppm Iron 72 59
ppm Copper 57.0 7.6
ppm Boron . 5.5 2.2
ppm Zinc 15 9
ppm Molybdenum 0.9 0.7
ppm Aluminum 1659 865

 

 

+ Values beyond the highest point of the curve.

154

Table ii. Emission Spectrograph Data of Diets Used in the
Eight Week Study

 

 

 

Basal + 5% Basal + 5%

Diets Basal Pectin N. F. Pectin L. M.
Elements

% phosphorus 0.624 0.646 0.582

ppm Sodium 2080 2016 2192

% Calcium 0.58 0.58 0.57

% Magnesium 0.05 0.05 0.05

ppm Manganese 18 16 12

ppm Iron 56 72 62

ppm Copper 15.7 19.5 21.5

ppm Boron 4.0 5.5 5.5

ppm Zinc 17 22 17

ppm Molybdenum 2.8 2.8 2.6

ppm Aluminum 50 60 44

 

 

155

Table iii. Weekly Protein Efficiency Ratios of the Basal
Fed Animals of Each Metabolic Period in the
Eight Week Study

u
I!

 

 

Groups
Period Two Weeks Four Weeks Six Weeks Eight Weeks
First week 5.254.06' 5.154.07 5.294.06 5.284.08

Second week 2.524.12 2.714.11 2.784.05 2.714.05

Third week 1.984.10 2.044.06 2.024.06
Fourth week 1.814.11 1.854.09 1.864.06
Fifth week 1.544.07 1.574.29
Sixth week 1.054.11 1.264.16
Seventh week 1.224.12
Eighth week 0.594.07

 

I
Standard error of mean.

156

 

 

 

Table iv. Weekly Protein Efficiency Ratios of the Pectin

N. F. Fed Animals of Each Metabolic Period in the

Eight Week Study

Groups

Period Two Weeks Four Weeks, Six Weeks Eight Weeks
First week 5.294.06' 5.014.97 5.164.07 5.204.05
Second week 2.624.07 2.524.12 2.554.07 2.604.11
Third week 2.104.08 1.904.10 1.964.11
Fourth week 1.654.08 1.644.11 1.554.15
Fifth week 1.564.11 1.424.11
Sixth week 1.054.11 1.084.11
Seventh week 1.124.08
Eighth week 0.024.17

 

'Standard error of mean.

157

 

 

Table v. Weekly Protein Efficiency Ratios of the Pectin L. M.
Fed Animals of Each Metabolic Period in the Eight
Week Study
Gropps -
Period Two Weeks Four Weeks Six Weeks Eight Weeks

 

First week 5.254.06' 5.054.09 5.174.05 5.074.06
Second week 2.544.08 2.654.09 2.684.07 2.724.09
Third week 2.064.05 1.974.06 1.784.09
Fourth week 1.724.11 1.664.08 1.754.10
Fifth week 1.584.05 1.574.10
Sixth week 1.024.12 1.044.17
Seventh week 1.074.10
Eighth week 0.554.14

 

'Standard error of mean.

158

 

 

 

 

Table vi. Emission Spectrograph Data from Livers of Animals
Fed Four Week-s~-~~~
Diet.

Basal + 5% Basal + 5%
Elements Basal Pectin N. F. Pectin L. M.
% Phosphorus 1.6924.08" 1.5624.05" 1.6594.07"
ppm Sodium 56564206 54474142 55424152
% Calcium 0.194.01 0.174.01 0.204.006
% Magnesium 0.0794.005 0.0754.002 0.0744.004
ppm Manganese 204.7 194.6 194.5
ppm Iron 485428 450415 597422
ppm Copper 29.945.5 26.041.2 25.04.9
ppm Boron 9.2 8.4 9.2
ppm Zinc 9545 9145 9645
ppm Molybdenum 0.954.06 0.924.08 0.994.06
ppm Aluminum 1556 1589 1506

 

'Extrapolated portion of curve.
"Standard error of mean.
+Values beyond the highest point of the curve.

Table vii.

159

Fed Six Weeks

Emission Spectrograph Data from Livers of Animals

 

 

 

 

Diet.

Basal + 5% Basal + 5%
Elements Basal Pectin N. F. Pectin L. M.
% phosphorus 1.5454.08' 1.4594.12' 1.5044.12'
ppm Sodium 50294512 52414251 54294198
% Calcium 0.204.01 0.174.01 0.194.01
% Magnesium 0.0714.002 0.0744.005 0.0824.004
ppm Manganese 204.9 184.7 194.6
ppm Iron 545450 471450 567445
ppm Copper 25.541.4 26.641.6 27.541.8
ppm Boron 9.94.8 8.14.45 8.84.5
ppm Zinc 9948 8844 9945
ppm Molybdenum 1.064.06 1.054.06 0.924.05
ppm Aluminum 1659 1659 1659

 

'Extrapolated portion of curve.
"Standard error of mean.
+Values beyond the highest point of the curve.

Table viii.

140

Fed Eight Weeks

Emission Spectrograph Data from Livers of Animals

 

 

 

 

Diet.

Basal + 5% Basal + 5%
Elements Basal Pectin N. F. Pectin L. M.
% phosphorus 1.4774.07' 1.5724.08' 1.7754.06'
ppm Sodium 29974111 27174125 5488475
% Calcium 0.184.01 0.154.01 0.214.01
% Magnesium 0.0704.002 0.0714.002 0.0794.002
ppm Manganese 1841.0 184.8 204.9
ppm Iron 567441 485455 614450
ppm Copper 21.041.2 22.541.0 24.44.6
ppm Boron 8.44.5 7.24.55 9.54.05
ppm Zinc 8442 8745 9644
ppm Molybdenum 1.074.09 .924.07 1.074.06
ppm Aluminum 1559 1659 1659

 

'Extrapolated portion of curve.
"Standard error of mean.

+Values beyond the highest point of curve.

Table ix.

141

Fed Six Weeks

Emission Spectrograph Data from Kidneys of Animals

 

¥
4—

 

 

 

Diet

Basal + 5% Basal + 5%
Elements Basal Pectin N. F. Pectin L. M.
% phosphorus 1.7224.055' 1.7594.055' 1.7844.044'
ppm Sodium 4527 4995 5020
% Calcium 0.214.01 0.224.01 0.214.01
% Magnesium 0.0794.001 0.0804.001 0.0804.002
ppm Mangesium 174.8 1941.0 184.6
ppm Iron 478452 512451 520444
ppm Copper 59.441.5 61.644.9 56.445.7
ppm Boron 9.741.6 9.141.0 8.740.7
ppm Zinc 9741.5 10445 9545
ppm Molybdenum 0.974.08 1.074.08 1.124.08
ppm Aluminum 1659 1659 1659

 

'Extrapolated portion of curve.
"Standard error of mean.
+Values beyond the highest point of the curve.

142

 

 

 

 

Table x. Emission Spectrograph Data from Kidneys of Animals
Fed Eight Weeks
Diet.

Basal + 5% Basal + 5%77
Elements Basal “Pectin N. F. Pectin L. M.
% phosphorus 1.6554.057' 1.6674.055' 1.2104.052'
ppm sodium 5120 5120 5120
% Calcium 0.224.02 0.214.01 0.254.01
% Magnesium 0.0774.001 0.0774.001 0.0774.001
ppm Manganese 2041.4 1841.0 194.7
ppm Iron 559456 474454 495454
ppm Copper 49.545.1 60.445.2 49.742.4
ppm Boron 10.14.8 9.64.9 9.14.4
ppm Zinc 9545 10445 9844
ppm Molybdenum 1.154.07 1.004.08 1.214.06
ppm Aluminum 1659 1659 1659

 

'Extrapolated portion of curve.
"Standard error of mean.
+Values beyond the highest point of the curve.

145

Table xi. Milligrams of Nitrogen Per Gram of Dried Liver
From Animals Fed Eight Weeks

 

 

 

 

Diet
Basal + 5%» Basal + 5%
Groups Basal Pectin N. F. Pectin L. M.
Two weeks 102.642.1 106.242.6 106.142.0
Four weeks 104.242.7 108.141.7 104.941.5
Six weeks 108.842.6 107.141.6 109.141.5
Eight weeks 107.142.1 106.645.1 111.741.6

 

'Standard error of mean.