L

PHYTATE AND ZINC EFFECTS ON THE CHROMIUM

NUTRITIONAL STATUS OF THE GROWING RAT

by

SHERRY ANN HENLEY, B.S.

A THESIS

IN

FOOD AND NUTRITION

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

IN

HOME ECONOMICS

Approved

I

1 Acc~pted I

August, 1983

' /' I ~-

~~ \ r· { I ~

ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to Dr. Barbara

Stoecker for her guidance, understanding, and patience during the

course of this research. I also wish to thank Dr. S. P. Yang and

Dr. Charles Garner for serving as committee members and providing

encouragement, advice, and suggestions on my research and education.

Support by U.S.D.A. competitive research grant # 59-2486-0-1-

480-0 and the Graduate School is greatly appreciated. I would also

like to thank Dr. Kathryn Keirn for technical assistance and advice

in the area of chromium research and animal care and surgery.

A very special thanks goes to my husband, Raymond Runyan, for

his scientific advice and expertise as well as his support and

patience. I am also grateful to my parents, Mr. and Mrs. Henley,

and to Mrs. Patricia Runyan for their encouragement and support

throughout my education.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS . . • • . . . . . • • • • • • • • • • • • • ii

LIST OF TABLES • • . . . . • • • • • • • • • • • • • • • • • vi

LIST OF FIGURES • • • • • • • • • • • • • • • • • • • • • • vii

I. INTRODUCTION • • • • • • • • • • • • • • • • • • 1

II. REVIEW OF LITERATURE • • • • • • • • • • • • • • • • 3

Chromium . . . . . . . . . . . . . . . . . . . 3

Chemistry • • • • • • • • • • • • • 4

Biochemistry and Mode of Action • • • • 5

Metabolism • • • . . . . • • • . . . • • 7

Physiological Role of Chromium • . • • • 11

Requirements and Nutritional Assessment • • • • • • • • • • . . . . . 14

• Phytic Acid • • • • • • • • • • . . . . . 16

Chemistry . . . . . . . . . . . . . . . 16

Biochemistry and Occurrence . . . . . . 17

Nutritional Implications . • • • • • 18

III. MATERIALS AND METHODS • • • • • • • • • . . . . 21

Experiment One . • • . . • • • • • • • • • 21

Animals and Diets • • • • • • • • • • • 21

IVGTT • • • • • • • • • • • • • • • • • 22

Glucose Determination • • • • • • • • • 22

Insulin Assay . . . . . . . . . . . . . 23

iii

IV.

Chromium Analysis

Phytate Analysis •

• • • • • • • • • • •

. . . . . . . • • • •

Experiment Two • • • • . . . • • • • • • • • •

Animals . . . . . . . . . . . . . . . . IVGTT • • • • • • • • • • • • • • • • •

Glucose Determination . . . . . . . . . Insulin Assay . .

Chromium Analysis

. . . . . . . . . . .

. . . . . . . . . . . Experiment Three • • • • • • • • • • • . . . .

Animals • • • • • • • • • • • • • • • •

OGTT •• . . . . . . . . • • . . . . • •

Glucose Determination . . . . . . . . . Insulin Assay . . . . . . . . . . . . . Chromium Analysis . . . . . . . . . . . Phytate Analysis • • • • • . . . . . . .

Statistical Analysis • • • . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . .

24

24

26

26

27

27

27

27

28

28

29

29

29

30

30

30

33

Experiment One • . • • • . . . • • . • . • • • 33

Growth Rates • . . . • • • . • • • . • • 33

Dietary Analysis •

Glucose and Insulin

iv

• • • • • • • • • • •

• • • • • • • • • •

33

33

Experiment Two • . • . . • • • • • • • . . . . Growth Rates • • • • • . . . . . . . . . Dietary Analysis • . . . . . . . . . . . Tissue Analysis . . . . . . . . . . . . Glucose and Insulin • • • • • • • • • •

Experiment Three . • • . . . . . . . . . . . . Growth Rates . • • • . . . . . . . . . . Dietary Analysis . . . . . . . . . . Glucose and Insulin . . . . . . . . . .

v. DISCUSSION . • • • • • • . . . • • • • . . . . . Growth Rates . • • • . . . . . • • • • • . . . Diet and Tissue Analysis • • . . • • • • • • •

Glucose and Insulin . . . . . . . . . . . . .

VI. SUMMARY AND CONCLUSIONS • • • • • • • • • • • • • •

REFERENCES • • . . . . . • • • • • • • • • • • • • • • • • •

APPENDICES • . . . . . . . . • • • • • • • • • • • • • • • •

A.

B.

PROGRAM SPECIFICATIONS USED WITH THE FLAMELESS ATOMIC ABSORPTION SPECTROPHOTOMETER . . . . . . . .

COMPOSITION OF MINERAL MIX . . . . .

v

34

34

34

34

35

35

35

35

36

51

51

52

53

57

59

66

67

68

LIST OF TABLES

1. COMPOSITION OF BASAL DIET FOR EXPERIMENTS ONE AN'D TWO • • . • . • • . • • • • • • • • • • • · 31

2. COMPOSITION OF BASAL DIET FOR EXPERIMENT THREE . . . . 32

3. PHYTATE AN'D CHROMIUM CONTENT OF DIETS FOR EXPERIMENT ONE . . . . . . . . . . . . . . . . . . . . . . . . 39

4. PLASMA GLUCOSE REMOVAL RATES FOR EXPERIMENT ONE 40

5. INSULIN VALUES FOR EXPERIMENT ONE . . . . . 41

6. CHROMIUM CONTENT OF DIET AND LIVER FOR EXPERIMENT TWO • • • • • • . . • . • • . • . • • . • · • • · 43

7 . PLASMA GLUCOSE REMOVAL RATES FOR EXPERIMENT TWO 44

8. GLUCOSE AN'D INSULIN VALUES FOR EXPERIMENT TWO . . 45

9. PHYTATE AN'D CHROMIUM CONTENT OF DIETS FOR EXPERIMENT THREE . . . . . . . . . . . . . . . . . . . . 47

10. GLUCOSE VALUES FROM OGTT FOR EXPERIMENT THREE 48

11. GLUCOSE INCREMENT FROM OGTT FOR EXPERIMENT THREE 49

12. INSULIN VALUES FROM OGTT FOR EXPERIMENT THREE . . . 50

vi

LIST OF FIGURES

1. Mean body weight for groups during Experiment One • . • . . . . . . • . . . . . . . . • . . . • . • 38

2. Mean body weight for groups during Experiment Two . . • . . • • . • • . . . . . . . . . . . • . . . 42

3. Mean body weight for groups during Experiment Three . . • . . . . . . . . . . . . . . . . . • . • • 46

•

vii

CHAPTER I

INTRODUCTION

The investigation of essential trace element requirements for

man and laboratory animals began in the seventeenth centurv with iron

and is continuing today with trace elements such as chromium.

Chromium, a subject of nutritional research since the 1950's,

has been found to be essential both for man and some laboratory

animals.

Chromium is involved in carbohydrate metabolism, with glucose

tolerance impairment being the main deficiency indicator. However,

chromium is also involved in protein and lipid metabolism.

Nutritional assessment of chromium status has been hampered bv

chromium analysis techniques and differences in bioavailability of

the various forms of chromium.

Some world-wide health problems have been related to decreased

bioavailability of minerals by binding to chelating agents such as

phytate. Phytate is found priMarily in grains and legumes and poses

the biggest problem in those populations consuming grains and legumes

as their staple diet. Therefore, the possibility of chromium being

bound and made unavailable by phytate is of concern to both the

scientific community and to the public sector.

The problem of phytate as a chelating agent for various trace

elements is still controversial as is the problem of trace element

interactions. The goal of this research was to assess the effect, if

1

2

any, of phytate or zinc on the chromium nutritional status of the

growing rat. Specific objectives include the following:

1. To produce and measure a chromium sufficiency and deficiency

as assessed by plasma glucose removal rates from an

intravenous glucose tolerance test and by glucose and

insulin curves from an oral glucose tolerance test.

2. To determine if sodium phytate added to a semipurified,

chromium deficient diet will bind remaining chromium and

further increase a chromium deficiency.

3. To determine if excess zinc, added to chromium deficient and

adequate diets, will affect chromium nutritional status as

measured by glucose removal rates and liver chromium

concentration.

CHAPTER II

REVIEW OF LITERATURE

Chromium

The immediate history of chromium research began in 1955 with

the observation by Mertz and Schwarz (1955) that rats fed a 30%

Torula yeast diet developed an impairment of glucose tolerance as

measured by intravenous glucose tolerance tests. Feeding vitamin E,

sulfur amino acids, factor 3, or selenium, all of which protect the

liver, did not prevent development of glucose intolerance (Schwarz

and Mertz, 1957). Addition of brewers' yeast to the diets, however,

did prevent the glucose intolerance and a glucose tolerance factor

was postulated (GTF) (Mertz & Schwarz, 1959). Chromium (III) was

identified as the component responsible for GTF's metabolic action

(Schwarz and Mertz, 1959).

Depletion of this essential trace element, chromium, has been

associated with impaired growth, decreased survival, fasting

hyperglycemia and glycosuria, elevated serum cholesterol, and aortic

plaques (Schroeder and Balassa, 1965; Schroeder, 1968 and 1969; Mertz

et al., 1974). Jeejeebhoy et al., (1977) indicated chromium

deficiency was also characterized by peripheral neuropathy, negative

nitrogen balance, and a decreased respiratory quotient.

Chromium is involved in protein, lipid and carbohydrate

metabolism as an essential element for optimal insulin activity in

all insulin-dependent systems tested (Roginski and Mertz, 1969;

Mertz, 1969). A decreased sensitivity of peripheral tissue to

exogenous or endogenous insulin resulted in "the hypothesis that

3

4

chromium acts as a cofactor in the initiation of peripheral insulin

action" (Mertz et al., 1974). Those groups at highest risk for

chromium deficiency have been identified as the aged, diabetics,

offspring or siblings of diabetics, pregnant women, and malnourished

children (Hambidge, 1974; Boyle et al., 1977; and Saner, 1980).

Chemistry

Chromium is a transition element in the first series and can

occur in every oxidation state from -2 to +6; however, the most

common states are ground state 0, +2, +3, and +6 (Mertz, 1967,

1969). Trivalent chromium is the most stable form of the element as

well as the most biologically active form (Mertz 1967, 1969).

Chromium(III) has a strong propensity to form coordination compounds,

complexes, and chelates (Mertz, 1967, 1969). Ligands coordinated

with chromium(III) have a very slow rate of exchange. Possible •

complexes include the following: water, ammonia, urea,

ethylene-diamine, halides, sulfate, and anions of various organic

acids (Mertz, 1967, 1969; Saner, 1980). The coordination number of

chromium(III) is 6 with an octahedron formation (Mertz, 1969).

Theoretically, slow ligand exchange would make chromium(III)

complexes more suitable in a structured role as a cofactor for

binding hormones to receptors or stabilizing proteins and less

suitable as active sites for enzymes which require a fast exchange of

ligands (Mertz, 1969). With time, alkaline conditions, and

temperature in the physiological range, coordinated compounds of

chromium(III) and water are hydrolyzed to hydroxo groups and form

polynuclear elated complexes (Mertz, 1967, 1969; Saner, 1980). This

5

process, referred to as alation, causes chromium complexes to

precipitate out of solution as chromium hydroxides and become

biologically ineffective (Mertz, 1967, 1969; Saner, 1980). However,

olation is prevented or reversed if chromium(III) compounds are

coordinated with stabilizing ligands (Mertz, 1969). Reversal of

alation is not likely under physiological conditions since the time

required is great.

Biochemistry and Mode of Action

The degree of biological activity of chromium(III) depends on

the type of compound in which it is found (Mertz, 1969; Saner, 1980).

There are two categories of compounds. The first includes simple

chromium compounds with chloro, aqua, or acetate groups or complexes

with amino acids, sugar, organic acids, or vitamins (Saner, 1980).

Natural chromium complexes found in plants and animals, including

brewers' yeast, fall into the second category and exhibit higher

biological activity than the first category of simple chromium

compounds (Saner, 1980). The second category of compounds is

referred to as "glucose tolerance factor" or GTF (Hertz et al.,

1974).

The structure of purified GTF prepared from yeast has been

postulated as a di-nicotinato, triamino acid, Cr(III) complex with

glycine, cysteine, and glutamic acid. Mass spectrometry was used as

the method of analysis (Toepfer et al., 1977). Synthetic GTF

compounds were formulated and found to have equal or better

insulin-potentiating activity in vitro than GTF frorn brewers' yeast

but thejr intestinal absorption was poor and placental transport was

6

lacking (Hertz et al., 1969, 1974). The synthetic GTF complexes did,

however, demonstrate more biological activity in vivo than simple

chromium compounds (Mertz et al., 1974). Synthetic GTF complexes

have been found to have different organ distribution than simple

chromium salts or GTF from brewers' yeast (Hertz and Roginski, 1971;

Mertz et al., 1974). Tuman and Daisy (1977) compared the effects of

GTF from brewers' yeast and synthetic GTF by injecting genetically

diabetic mice intraperitoneally with one of the two chromium

compounds. A reduction of high blood glucose levels, triglycerides,

and cholesterol were measured in both the synthetic and natural GTF

groups. The synthetic GTF, however, had a much more rapid onset of

blood glucose lowering effects than did GTF from brewers' yeast

(Tuman and Daisy, 1978).

Schroeder et al. (1963) tested the effect of chromium, cadmium

and lead on the growth and survival of laboratory animals to evaluate

toxicity and essentiality effects. The same study indicated that

simple chromium(III) salts (5 ppm) added to the drinking water of

male rats fed a low chromium diet increased growth rates, mature

weights and survival rates up to 510 days.

Several in vitro studies using epididymal fat tissue from rats

indicated that chromium significantly increased the effect of insulin

on the incorporation of glucose into the cell (Nertz et al., 1961;

Mertz and Roginski, 1963; Campbell and Mertz, 1963). Another in

vitro studv using polarographic techniques suggested that chromium,

as part nf a ternary complex, initiated the formation of disulfide

linkages between the intra-chain disulfide of insulin and

7

sulfhydryl groups of the mitochondrial membrane (Christian et al.,

1963). More information was obtained from additional in vitro

studies using D-galactose, a nonutilizable sugar requiring insulin

for cell uptake, as a substrate. The rate of entry of the sugar into

rat epididymal fat pad was only slightly increased with insulin but

greatly increased with insulin and chromium and it demonstrated dose

response characteristics (Mertz and Roginski, 1963; Mertz, 1967).

Similar effects were shown during other in vitro experiments using

glucose transport into isolated rat lens (Mertz, 1979) and

insulin-dependent mitochondrial swelling (Campbell and Mertz, 1963).

Metabolism

Chromium metabolism differs from that of other trace minerals

because of its dependence on the chemical form of the element (Mertz,

1976; Saner, 1980). Studies on chromium metabolism involve

intestinal absorption, transportation within the body, tissue levels

and distribution and excretion.

Absorption of an oral dose of trivalent chromium salts bv rats

fell within the range of 0.5 to 3.0% (Visek et al., 1963; Mertz, et

al. 1965; Mertz, 1969; Mertz and Roginski, 1971) whereas normal human

subjects were found to absorb 0.3 to 1.3~ of an oral dose of chromium

salts (Donaldson and Barreras, 1966; Saner, 1980). Research with

GTF, however, indicated that 10-25r of GTF chromium from brewers'

veast was absorbed (Mertz, 1975; Saner, 1980). Absorption of

chromium(III) from orally administered physiological doses is verv

low regardless of the amount of dose given or chromiuM nutritional

status (Mertz, 1965, 1967; Toepfer, 1977). The low absorption rate

8

is partially attributed to the tendency of chromium(III) salts to

elate in neutral or basic solutions such as those found in the small

intestine (Donaldson and Barreras, 1966; Visek et a1., 1953).

In vivo and in vitro studies with rats, using chelating agents,

found that the primary absorption site of chromium was in the jejunum

or mid section of the small intestine with Jesser amounts absorbed in

the ileum and duodenum (Chen et al., 1973). A number of substances

influence the absorption of chromium. Those that depress chromium

absorption include: zinc, iron, manganese, calcium, and titanium.

Glutamate and penicillamine enhance the absorption of chromium (Mertz

and Roginski, 1971). Zinc apparently competes for the same

absorptive sites in the intestional mucosa as chromium. Hahn and

Evans (1975) found a decreased absorption and intestinal mucosal

f 51c h d · · d · h · · d f · · content o r w en a m1n1stere w1t z1nc to z1nc- e 1c1ent

animals. In the same study there was a decreased absorption and

intestinal mucosal content of 65 zn when administered with chromium to

zinc-deficient animals (Hahn and Evans, 1975). Since chromium and

zinc were also eluted in the same mucosal supernatant fraction from

animals in the above experiment, the authors hypothesized that the

same ligand may bind both metals causing competition in absorption

(Hahn and Evans, 1975).

Once absorbed chromium(III) is bound to transferrin,

a S-globulin, which also transports iron (Hopkins, 1964). Of the 2-3

mg of transferrin per ml of serum, 30% is saturated with iron and the

remainder is available for chromium(III) and other trace mineral

transport (Hopkins, 1964). The mechanism for control of cellular

chromium transport has not been established; however, Lifschitz et

al. (1980) have postulated that thyroid hormone may play a role in

chromium transport. Their study with 51cr and parathyroidectomized

rat h d · f d · f · 51c · · h s s owe un1 orm epress1on o t1ssue:serum r rat1os w1t

complete reversal on replacement with thyroxin (Lifschitz et al.,

1980).

The placental transfer of simple chromium salts vs. brewers'

yeast chromium was studied in the rat (Mertz et al., 1969).

9

Placental transport of chromium(III) was found to occur only with GTF

as opposed to 2 ppm chromium salts added to drinking water (Mertz et

al., 1969).

Hopkins investigated the distribution of physiological amounts

of injected 51crc13

and observed that on the fourth day after

administration spleen, kidney, testis, and epididymis retained the

highest percentage (104-200%) and heart, lung, pancreas, and brain

retained the least (10-31%) with other tissues having intermediate

values (Hopkins, 1965). 51

chromium(III) has a rapid blood clearance,

whereas other organs retain 51chromium(III) much longer, which

suggests there is no equilibrium between tissue and blood chromium

levels (Hopkins, 1962). The highest concentration of chromium, given

as GTF from brewers' yeast, was found in liver tissue which suggests

that the liver may be the site of the specific pool of biologically

important chromium in animal or man (Mertz and Roginski, 1971).

Jain et al. (1981) recently found liver 51

chromium in the rat to be

SO to 100% exchangeable, further supporting the liver as a

physiological poo] and regulator of chromium metabolism. As mentioned

10

previously, there was a different tissue distribution for

chromium(III) salts and GTF chromium with onlv the GTF form

concentrating in the liver.

Chemical analyses of postmortem tissues for chromium (Schroeder

et al., 1962) revealed a decreasing tissue chromium concentration

with increasing age in the U.S. Tissue chromium concentrations of

adults in the U.S. were much lower than those in the Far East, Mid

East and Africa (Hambidge, 1974) as well as lower than in some wild

animals (Schroeder, 1968; Hambidge, 1974).

Chromium administered either orally or intrRvenously is excreted

primarily by the kidney with only small amounts being excreted

through the feces from bile or through the skin (Hopkins, 1965;

Donaldson and Barreras, 1966; Donaldson and Rennert, 1981). There

was an exponential decrease with time in renal clearance of

intravenously administered 51crc13

(Donaldson and Rennert, 1981).

Assessment of urinary chromium excretion under specifically

controlled conditions may reflect the chromium status of an

individual (G~rson and Saner, 1978). Schroeder (1968), Gurson and

Saner (1978) and Rabinowitz (1980) all reported an increase over

fasting of urinary chromium concentration following an oral glucose

challenge in normal adult humans. In contrast, Davidson et al.

(1974) and Vanderlinde et al. (1979) reported either no change or a

decrease in urinary chromium concentration following a glucose

challenge. Gtirson and Saner (1978) have observed a significant

positive relationship between urinary chromium and creatinine

concentration and recommend the use of the Cr/Cre ratio as a valuable

11

criterion in the evaluation of chromium nutrition. Anderson et al.

(1983), however, recently reported that urinary chromium excretion

may not be a good indicator of chromium nutritional status but that

it appeared to be a good indicator of chromium intake. The same

studv also suggested that absorption of supplemental inorganic

chromium was similar to the absorption of chromium from normal

dietary sources (Anderson et al., 1983).

Physiological Role of Chromium

Chromium deficiency may cause insulin resistance,

hyperinsulinemia, impaired glucose tolerance, and hyperlipidemia all

of which were reversed by chromium supplementation (Riales and

Albrink, 1981). Early studies demonstrated the effect of a glucose

challenge on both chromium deficient and supplemented animals as

measured by glucose tolerance tests (Schwarz and Mertz, 1959) with a

reversal of symptoms in deficient animals when chromium was added

at 2 or 5 ppm chromium(III) in the drinking water (Underwood, 1977).

Current research, however, is involved with evaluating serum chromium

response to a glucose load by normal and chromium deficient subjects.

For example, Rabinowit~ et al. (1980) reported that in most normal

subjects, plasma chromium levels increased following a glucose

challenge, along with Liu and Morris (1978) who reported an increase

in serum chromium but only after supplementation with GTF chromium.

Davidson and Burt (1973) and Pekarek et al. (1975), however, found

that serum chromium concentration fell in response to either an oral

or intravenous glucose challenge. Vanderlinde et al. (1979), using

the most current ultratrace techniques such as neutron activation or

12

flameless atomic absorption spectroscopy, found no significant change

between fasting and two hour post prandial serum chromium

concentrations in normal subjects with values at the very edge of

detection limits.

In vitro and in vivo chromium deficient systems have shown a

.decreased glucose uptake in response to doses of insulin. Higher

doses of insulin are required by chromium deficient systems to obtain

responses si~ilar to those of chromium sufficient controls

(Underwood, 1977). On the other hand there is no difference in

glucose uptake response between groups in the absence of insulin,

which suggests that an increased plasma chromium response to the

ingestion of glucose is an indirect one that occurs through

stimulation of insulin secretion (Mertz et al., 1974). These results

may suggest two possibilities concerning plasma chromium levels after

fasting and in the fed state: 1) an increased plasma chromium

increment following a glucose or insulin load indicates an adequate

chromium body pool; 2) lack of an increment may indicate a deficient

chromium body pool (Mertz et al., 1974).

Glucose tolerance impairment, as a symptom of kwashiorkor,

has in some cases responded dramatically to chromium supplementation,

as seen in those cases of infant protein-calorie malnutrition in

Jordan, Nigeria, and Turkey (Hopkins et al., 1968; Gurson and Saner,

1971). Malnourished infants in Egypt, however, showed no effect from

chromium supplementation and their tissue chromium levels were normal

indicating their glucose intolerance was not due to a chromium

deficiency (Underwood, 1978).

13

Two patients on long-term total parenteral nutrition exhibited

glucose intolerance, neuropathy, weight loss and low hair

concentration~ all of which responded to supplementation with either

150 ~g or 250 ug of chromium as Crc13

daily (Jeejeebhoy et al., 1977;

Freund, 1979).

Recent studies with elderly and non-insulin dependent diabetic

subjects, using chromium-rich brewers' yeast in the experimental

group and Torula yeast as a control elicited improved glucose

tolerance and decreased total lipids in the brewers' yeast group for

both elderly and diabetic subjects (Offenbacher and Pi-Sunyer, 1980).

Insulin output also decreased with improved glucose tolerance which

indicates these elderly people may have had a marginal chromium

deficiency that could be reversed with a chromium-rich source such as

brewers' yeast (Offenbacher and Pi-Sunyer, 1980). Other studies with

elderly or diabetic subjects have achieved similar results with

repletion requiring greater lengths of time than in the infant PCM

patients (Underwood, 1977).

Mertz (1979) strongly recommends that chromium supplementation

studies include measurement of effect on insulin levels since insulin

may be more directly related to chromium response than glucose. Liu

and Abernathy's (1982) recent results support this theory. In a

group of college subjects, they found the group with low insulin

levels had a significantly higher relative chromium response

(RCR is defined as 1-hour serum chromium level/fasting serum chromium

level x 100) than did the group with high insulin levels (Liu and

Abernathy, 1982). The higher RCR in the low insulin group indicated

14

normal glucose tolerance and supported the use of the RCR as an

assessment tool for chromium nutritional status (Liu and Abernathy,

1982).

R~quirements and Nutritional Assessment

The Food and Nutrition Board of the National Academy of Sciences

has not established a Recommended Dietary Allowance for chromium, but

it has listed an estimated Safe and Adequate Intake as 50-200 pg per

day (Food and Nutrition Board, 1980). Available data (Toepfer et

al., 1973; Wolf et al., 1974; Kumpulainen and Koivistoinen, 1977) on

total chromium content of foods may not be nutritionally valuable due

to recent advances in chromium analysis methodology and to differing

bioavailability of the various forms of chromium (Underwood, 1977).

Toepfer et al. (1973), using the glucose oxidation procedure in

the presence of rat epididymal tissue and insulin, found no

significant relationship between total chromium and biologically

active chromium in food. Chromium content of foods has been shown to

decrease with processing, especially in sugar and flour (Wolf et al.,

1974).

Previous data on serum and urine chromium concentrations have

been revised with the use of more advanced ultratrace techniques.

For example, in 1966 human serum chromium concentration was reported

as 23 ng per ml whereas current values are in the range of 0.1 ng per

ml (Donaldson and Rennert, 1981). The range for 24 hour urinary

chromium excretion was reported as 150 ~g per day (Underwood, 1977)

in 1966 and as 0.22 pg per 24 hours in 1983 (Anderson et al., 1983).

15

Evaluation of chromium nutritional status in man is difficult

due to the inadequacy of analytical methods (Saner, 1980). The

intravenous glucose tolerance test has been used to measure chromium

deficiency in laboratory animals on controlled diets as

expressed by glucose removal rate after administration of a glucose

load (Mertz et al., 1965), but the test is valid onlv under -- .

controlled laboratory conditions since response to glucose load could

be an indicator of disease symptoms as well as other trace element

deficiencies.

Plasma chromium levels do not accurately reflect chromium

status because plasma chromium is probably in equilibrium with the

physiological chromium pools only in response to elevated insulin

levels in the blood (Mertz, 1979).

The diagnosis of inadequate chromium stores is promoted by

simultaneous findings of elevated insulin levels and responses, or

glucose tolerance impairment, or both (Mertz, 1979). Chromium

supplementation of individuals with little or no chromium response

who subsequently demonstrate an increased chromium response to a

glucose load further supports diagnosis of inadequate chromium stores

(Mertz, 1979). Liu and Abernathy's (1982) RCR is a possible tool for

use in assessing chromium nutritional status.

Due to a high consumption of refined carbohydrates in this

country, it has been postulated that the American diet contains

inadequate amounts of chromium (Schroeder, 1968; Underwood, 1977).

The current estimate for chromium content in Western diets is 50 to

16

100 ~g/day, with some otherwise adequate diets supplying only

5 ~g/day (Food and Nutrition Board, 1980).

Phytic Acid

Phytates from plants represent a complex class of naturally

occurring compounds that can significantly influence the functional

and nutritional properties of foods, especially trace mineral

bioavailability (Erdman and Forbes, 1977; Davies, 1979; Maga, 1982).

Phytate is an organic factor which can bind essential minerals to

form insoluble and indigestible complexes in the lumen of the

intestinal tract ·and prevent their absorption (Davies 1979; Erdman

and Forbes, 1977). Increased awareness of certain health problems,

such as diverticulosis, colon cancer, elevated blood cholesterol

levels, and increased insulin resistance and/or glucose intolerance

in adult onset diabetes, has led to an increased emphasis on

consumption of whole grains and legumes, which are high in natural

fiber and phytate (Davies, 1979). Rising prices of animal protein

have also influenced consumption of grains and legumes.

Chemistry

Phytic acid (myo-inositol 1,2,3,5/4,6-hexakis dihydrogen

phosphate) is the chief storage form of phosphorus in all plant seeds

with natural legumes and cereal grains having the highest

concentrations (Oberleas, 1983; Maga, 1982). At a pH of 7.4, in

vitro, the phytate ion bound metals in the following decreasing

++ ++ ++ ++ ++ +++ ++ order: Cu , Zn , Ni , Co , Mn , Fe , and Ca (\rohra et al.,

1965). Of the phytate-mineral complexes tested, ferric ion was least

17

soluble in acid, zinc was least soluble in slightly acid solutions,

and the remaining five were least soluble in slightly alkaline

conditions (Oberleas, 1983). The chemical structure of phytic acid

is continually questioned due to the many isomeric forms of the

phosphate groups within the compound (Erdman and Forbes, 1977).

Biochemistry and Occurrence

In legumes, phytate is distributed uniformly throughout the

plant and is associated with the protein, while in cereal grains, it

is contained in both the bran and germ (Maga, 1982; Oberleas, 1983).

Corn, however, contains phytate primarily in the germ (Maga, 1982;

Oberleas, 1982). Several other types of food items have also been

analyzed for phytate content. Essentially no phytate was found in

bananas, celery, citrus fruits, lettuce, mushrooms, onions, or prunes

with trace amounts in apples, broccoli, carrots, and green beans

(Harland and Harland, 1980). Artichokes, figs, potatoes, and

strawberries contained moderate amounts, while cereals, legumes and

nuts contained the highest concentration of phytate (Harland and

Harland, 1980). Corn, rice, and wheat had a phytate concentration of

approximately 1%, while defatted sesame meal was found to contain

from 1.4 to 5.2% phytate (Erdman and Forbes, 1977; Maga, 1982).

Phvtate in wheat, rice, brood beans, castor, and sesame occured

mainly as mixed potassium-magnesium salts associated with protein in

the kernel (Erdman and Forbes, 1977; Reddy et al., 1980). Soybeans,

however, contained phytate as a calcium-magnesium-potassium salt

(Reddy~~., 1982).

18

Processes for removing phytate from soy protein have been

developed but are not yet used commercially (Hartman, 1979). Reddy

et al. (1982), in their review and discussion of methods for removal

of phytate, suggest that methods are not yet feasible on a commercial

scale. Methods commonly used in food processing form insoluble

protein-phytic-acid-mineral complexes (Forbes et al., 1979).

Phytic acid is not degraded by normal cooking procedures;

however, heating in acid or alkaline solutions will degrade the

molecule (Maga, 1982; Oberleas, 1983). Phytase, a natural component

of cereal grains, does faciltate the breakdown of phytate (Oberleas,

1983). Hydrolysis of phytate by phytase has been shown in yeast

leavening, with both added and natural phytase (Oberleas, 1983).

Studies of phytate content in leavened and unleavened bread have

correlated clinical zinc deficiency with consumption of unleavened

bread in Middle Eastern countries (Oberleas, 1983).

Nutritional Implications

Monogastric animals such as man have a very low phytase activity

in the digestive tract and, therefore, are not able to completely

cleave certain minerals from the phytate complex (Erdman and Forbes,

1977). Research on phytate-chromium interaction indicated that

phytate significantly decreased chromium transport through the rat

intestine both in vitro and in vivo (Chen et al., 1973). Fasted - - --animals, in the above study, absorbed chromium faster than did the

nonfasted animals (Chen et al., 1973). Davies and Nightingale (1975)

demonstrated a similar effect measuring zinc instead of chromium.

19

One study, using a 1% phytate diet, showed inhibition of growth and

decreased absorption of iron, copper, manganese, and zinc whether the

diet was supplemented or unsupplemented with zinc (Davies and

Nightingale, 1975).

Phytate-zinc interaction studies have indicated: 1) a high

calcium diet with phytate decreased the bioavilability of zinc

(Morris and Ellis, 1980; Nahapetian and Young, 1980); and 2) a

phytate to zinc molar ratio of 10 or less is optimum for humans

(Oberleas and Harland, 1981). Reduction of zinc bioavailability is

the most critical problem associated with phytic acid; however,

decreased iron utilization has also been shown by some, but not all,

researchers (Maga, 1982). Not all studies with iron and phytate,

though, haveshown decreased iron availability (Maga, 1982). Animal

studies with iron and phytate indicate a high biological availability

of monoferric phytate, the major form of iron in wheat (Morris and

Ellis, 1976). Hunter (1981), using sodium phytate up to 4% of the

diet, showed no difference in iron absorption between controls and

iron deficient rats but did suggest that effects of phytate may

depend on method of administration as well as length of time of

phytate administration.

Fiber may have an independent role from that of phytate in

binding minerals and making them unavailable for absorption

(Nahapetian and Young, 1980; Godara et al., 1981). The addition of

7% bran (12 mg phytate/roll) to wheat bread decreased iron absorption

by a factor of 2 (Bjorn-Rasmussan, 1974). Reinhold et al. (1975);

Ismail-Beigi et al. (1977) and Maga (1982) demonstrated that fiber

·zo

produces a separate or even additional effect to that of phytate;

totally dephytinized whole Meal bread and bran showed an enhanced

ability, compared to non-dephytinized bread, to bind calcium, zinc

and iron. Purified cellulose, without phytate, added to a purified

diet resulted in significant decreases of serum calcium, inorganic

phosphorus, and iron levels (Godara et al., 1981).

CHAPTER III

MATERIALS AND METHODS

Experiment One

Animals and Diets

Fifty 21-day-old, male Sprague-Dawley rats were fed variations of

the 30% Torula yeast low chromium diet outlined in Table 1. All

diets contained 12.5% digestible protein. 1 Sodium phytate was added

to the diet of one group at 1.38% (0.8% phytate) and to another group

at 2.08% (1.2% phytate). Two parts per million chromium as chromic

chloride (CrC13

·6H20) was added to the diet used as the adequate

chromium/Torula yeast diet. A second adequate chromium diet was made

2 by substituting brewers' yeast for Torula yeast in the basal diet.

Added phytate and chromium replaced sucrose in the basal diet.

Sodium content was equivalent in all diets, based on total amount in

sodium phytate.

Once a week all animals were given the basal diet without

phytate to which was added 2.5 times the magnesium, manganese, iron,

zinc, and copper contained in the regular diet. The animals were

fasted for 12 hours overnight, then given 20 g of the mineral

supplemented diet without phytate for 24 hours. At the end of the 24

hours, any remaining food was then removed and the regular diet

refed.

1sigma Chemical Company, St. Louis, MO.

2united States Biochemical, Cleveland, OH.

21

•

22

All animals were housed individually in plastic shoe-box cages

Rnd fed ad libitum from ceramic feed cups. Distilled, deionized

water in glass bottles with glass sipper tubes was provided. Animals

were weighed weekly.

IVGTT

Intravenous glucose tolerance tests (TVGTT) were performed

during weeks 5, 8, and 14 of the study. After a 14 to 16 hour fast,

50 mg glucose per 100 g body weight as a 25% glucose solution was

injected into the tail vein of the unanesthesized animal using a 27

gauge, ~ inch needle. Restraining cages were used to hold the

animals during glucose injection and blood collection. Blood was

collected from the tip of the tail in heparinized, 1 ~1 capillary

tubes immediately before (0 time) and 10, 20, 30, and 40 minutes

after the glucose load. The blood was centrifuged and the plasma

removed immediately after blood collection to be analyzed for glucose

on the same day.

Glucose Determination

The glucose in 20 ~1 of plasma was determined by a coupled

1 enzymatic colorimetric procedure . A 4 ml aliquot of a mixture of

horseradish peroxidase (E.C. 1.11.1.7) and glucose oxidase (E.C.

1.1.3.4) enzymes and o-dianisidine was added to the serum. The

following coupled reactions produced a brown color (oxidized

0 -dianisidine) proportional to the original glucose concentration:

1Technical bulletin 510, Sigma Chemical Company~ St. Louis.

23

1. Glucose oxidase Gluconic Acid + H

2o2

2. Peroxidase Oxidized o-Diansidine

After 45 minutes, absorption at 445 nm was measured in a

spectrophotometer.

Glucose removal rates were determined from the glucose tolerance

tests. The log of the increment above fasting was calculated against

time and the formula

log cl c2 K = X 2.3

t2 tl

was applied, where K is a constant and C is the blood glucose

increment value at time t (Duncan, 1956).

Insulin Assay

Insulin response to a glucose load was measured during week 15

of the study using the same procedures for blood collection used for

the IVGTT. However, blood was drawn only at 10 minutes after fasting

for the insulin assav. A radioimmunoassay (RIA) kit1

was utilized to

measure plasma insulin. Samples o~ the unknown and standard insulin

solutions were incubated for 6 hours with suspended insulin binding

reagent. 125 I-labelled insulin was added and the samples incubated

for an additional 18 hours. The insoluble antigen-antibody complex

was separated from the soluble free insulin by centrifugation.

Radioactivity was counted in the precipitate for both standards and

unknowns.

1Arnersham Corporation, Arlington Heights, TL.

24

Chromium Analysis

Chro~ium content of the diets was determined by flameless atonic

1 absorption spectrophotometry . Samples (150 mg) were weighed into

borosilicate glass tubes and placed in a sealed coil muffle furnace.

The temperature was raised 50°C per hour starting at 100°C up to

300oC; the temperature was then set at 500°C for 24 hours. The

samples were then removed from the muffle furnace and cooled. 50 wl

of H2o2 , distilled H20, and concentrated nitric acid were added to

samples and blanks and allowed to evaporate to dryness in a dry bath

at 100°C. Samples were then returned to the muffle furnace and ashed

at 500°C until all samples were white. After removing from the

muffle furnace and cooling, samples were diluted with lN hydrochloric

acid and analyzed on the flameless atomic absorption

spectrophotometer. All samples were measured in a linear range of

0-10 ppb chromium. Samples above that range were diluted with 1 N

HCl to the appropriate range. Chromium standards of 2 and 5 ppb were

made from a Certified Atomic Absorption Standard Reference Solution2

in 1 N hydrochloric acid. Program specifications for the

instrument are listed in the Appendices.

Phytate Analysis

An ion-exchange method for phytic acid analysis, developed by

Ellis and Harris (1982) as an improvement of the Harland and Oberleas

1Perkin-Elmer Zeeman 5000 with HCA 400 Programer, Perkin Elmer Corp., Norwalk, Conn.

? -Fisher Scientific Company, Fair Lawn, N.J.

25

method (1977), was utilized with one modification. Samples (2 g) of

dried material were extracted for 2 hr at room temperature with 40 ml

of 2.4% HCl. The mixture was centrifuged and then vacuum filtered

through Whatman No. 41 filter paper. The filtrate was refrigerated

for no more than 1 week. The 0.7 x 15.0 em columns were packed with

0.5 g of 200-400 mesh AG1-X8 anion exchange resin (chloride form) in

a deionized water slurry. Before use, the columns were washed with

10 rnl of 0.7 M NaCl to assure chloride saturation of the resin and

then with two column volumes of deionized water. One ml of the

sample extract was mixed with 1 ml of EDTA-NaOH solution, diluted to

30 ml with deionized water, and applied to the column. The sample

container was rinsed, and the column was washed with 15 ml of

deionized water to elute most of the inorganic phosphate. The

remaining inorganic phosphate was eluted with 15 ml 0.1 M NaCl. The

phytate was then eluted from the column with 15 ml of 0.7 M NaCl and

collected in a 100-ml micro-Kjeldahl flask. To the digestion flask,

was added 0.5 ml of concentrated H2so4 , 3.0 ml concentrated HN03

and

two glass beads. The mixture was digested on mediu~ heat until

active boiling ceased and a cloud of thick yellow vapors appeared in

the neck of the flask. The flasks were heated for 5 more minutes on

medium heat, 5 minutes on low heat, and then allowed to cool.

Deionized water (15 ml) was added, and the flasks were placed in a

boiling water bath for 15 min. The contents of the digestion flask

were quantitatively transferred to a 50 ml volumetric flask. The

phosphorus in the digest was determined by a modification of the

Fiske-Subbarow (19~5) method. The digest was diluted to about 35 ml

26

1 with deionized water, then 2 ml of molybdate solution were added and

the contents mixed. Next 1 ml of sulfonic acid reagent was added and

the 50 Ml flask was made to volume with deionized water. After

standing 10 to 15 minutes, the color was read at 640 nm on a Bausch

and Lomb spectrophotometer. The phytic acid values were calculated

from the organic phosphorus by assuming that one molecule of phytic

acid contained six molecules of phosphorus (1 mg phytate = 28.2% P).

Experiment Two

Animals

Thirty weanling, female Sprague-Dawley rats from dams maintained

on a low chromium, Torula yeast based diet were used in this second

experiment. The animals were fed the 30% Torula yeast low-chromium

diet outlined in Table 1. All diets contained 12.5% digestible

protein. Five parts per million chromium as chromic chloride was

added to the diets of the adequate chromium groups. Zinc

concentration was doubled (compared to the NAS-NRC recommendations)

in the diet of one low chromium group. Both copper and zinc were

doubled in one low chromium and one adequate chromium group.

The animals were housed individually in suspended galvanized

mesh cages and fed ad libitum from ceramic feed cups. Distilled,

deionized water in glass bottles with glass sipper tubes was

provided. Animals were weighed once weeklv.

1Modification: The molvbdate solution was acid stabilized as in the original method by Harland and Oberleas (1977).

27

IVGTT

IVGTT were performed during weeks 4 and 8 utilizing the

procedures described for Experiment One.

Glucose Determination

The glucose in 10 ~1 of plasma was determined using the Beck~an

Glucose Analyzer 2 with an Oxygen Electrode. The sample volume was

manually pipetted into enzyme reagent 1 in a cup containing an

electrode responsive to oxygen concentration. 8-D-glucose from the

sample combined with dissolved oxygen from the solution, according to

the reaction:

8-D-Glucose + 02

glucose oxidase 1

. . d 0 G ucon1c ac1 + H

2 2

In the reaction, oxygen was consumed at the same rate as glucose

reacted to form gluconic acid. The instrument provided a direct

readout of mg glucose per 100 ml. Glucose removal rates were

determined with the same formula as applied in Experiment One.

Insulin Assay

Plasma insulin levels were analyzed by radioimrnunassay during

week 9 of the study as described under Experiment One.

Chromium Analysis

Diets were analyzed for chromium concentration utilizing the

same procedure described under Experiment One.

1Glucose oxidase reagent, Fisher Scientific Co., Fair Lawn, N.J.

28

Liver chromium concentration was also analyzed using a similar

procedure as for diets, with several additional treatments. After

sacrifice, the liver was removed, rinsed in distilled water and

frozen immediately. Using only glass instruments for slicing the

tissue, samples (250 mg) were weighed into new borosilicate glass

tubes and freeze dried for 24 hours. The samples were re-weighed

and ashed in the muffle furnace as previously described.

Experiment Three

Animals

Forty-eight, weanling male Sprague-Dawley rats were fed a 32%

Torula yeast low chromium diet as outlined in Table 2. All diets

contained 13.3% digestible protein. Sodium phytate was added to the

diet of a low chromium and of an adequate chromium group. Sodium

content was equivalent in all diets. Extra zinc was added to the

mineral mix to equal 129 mg Zn per kg diet and a phytate:zinc molar

ratio of 8:1. Two parts per million chromium as chromic chloride was

added to the diet for the adequate chromium groups. Added phytate

and chromium replaced sucrose in the basal diet. Once weekly (~4

hours) starting week 4, the phytate groups were fed equivalent but

non-phytate containing diets.

Animals were housed in pairs in plastic-shoe-box cages. Animals

were fed ad libitum except for the non-phytate groups which were

restricted to 12 g a day per animal during week 4 and the first 3

days of week 5. Distilled water in glass or plastic bottles with

glass sipper tubes was provided on all cages. Animals were weighed

once weekly.

29

OGTT

An oral glucose tolerance test was performed during week 6.

After a 14 to 16 hour fast, 200 mg glucose per 100 g body weight in a

50% glucose solution was intubated into the stomach using an 18 gauge

intubation needle. Each animal was wrapped loosely in a small towel

with the tail free and held by the researcher with one arm, leaving

the other free to collect blood from the tail veins. Blood was

collected from the tip of the tail in EDTA-coated 1 ml capillary

tubes immediately before (0 time) and 30, 60, and 120 minutes after

the glucose load. The blood was centrifuged, and the plasma was

removed immediately and frozen for insulin and glucose

determinations.

Glucose Determination

Plasma glucose was determined using the Beckman Glucose Analyzer

as described for Experiment Two.

Insulin Assay

Another RIA kit 1 , found to have improved precision compared to

the kit used in Experiment One, was utilized for the insulin assay.

All plasma samples analyzed for glucose were also analyzed for

insulin. Samples of the unknown, standards, and control solutions

Here incubated for 16 hours at 4°C with specific anti-insulin

antiserum and 125I-labelled insulin. Bound and free radiolabeled

insulin were separated by adding double antibody and polyethylene

1cambridge Medical Diagnostics, Inc., Billerica,~~.

30

glycol, which precipitated the antigen-antibody complexes. Samples

were incubated for 10 min at room temperature and centrifuged, and

the precipitate was counted. By counting the radioactivity in the

precipitate for both standards and unknowns, a standard curve was

constructed and values were calculated for all samples.

Chromium Analysis

The same method was employed to analyze the diets for chromium

content as described under Experiment One.

Phytate Analysis

Diets were analyzed for phytate content as described under

Experiment One.

Statistical Analysis

The Statistical Analysis System (S.A.S.) was used to determine

basic statistics (mean, standard error of the mean, range and

variance) and to compare differences among group means. A general

linear model (GLM) and a Duncan's Multiple Range Test were also

utilized at the 0.05 alpha level to determine significant differences

among groups. A repeated measures design was done crossing dietary

group with time to determine the differences within groups over time

for glucose tolerance.

31

TABLE 1

COMPOSITION OF BASAL DIETl FOR EXPERI'!-'fENTS

ONE AND TWO

Ingredient % of diet

2 Torula yeast

Methionine 3

4 Sucrose

Corn oil5

Hineral mix6

Vitamin mix (AIN 76A) 7

NaC18

Choline bitartrate9

30.00

0.40

58.67

5.00

3.50

1.00

1.32

0.11

1Meets NAS-NRC recommendations for growth of weanling rats.

2rorula dried yeast, Teklad Test Diets, Harlan Sprague Dawley, Inc., Madison, WI.

3DL-methionine, United States Biochemical Corp., Cleveland, OH.

4 Granulated sugar, Topco Associates, Inc., Skokie, IL.

5 Mazola, Best Foods, Englebrook Cliffs, NJ.

6Hineral mix, formula based on AIN mineral mixture 76, without sodium chloride and chromium potassium sulfate (Appendix A).

7AIN vitamin mixture 76A, United States Biochemical Corp, Cleveland, OH.

8 sodium chloride, MCB Manufacturing Chemists, Inc., Cincinnati, OH.

9r.holine bitartrate, United States Biochemical Corp. Cleveland, OH.

32

TABLE 2

COMPOSITION OF BASAL DIETl FOR EXPERIMENT THREE

Ingredient

2 Torula yeast

M h . • 3 et 10n1ne

4 Dextrose

5 Cornstarch

Corn oil6

Mineral mix7

Vitamin mix8

Choline bitartrate10

% of diet

32.10

0.40

30.00

26.60

5.00

3.50

1.00

1.29

0.11

1Meets NAS-NRC recommendations for growth of weanling rats.

2Torula dried yeast, Teklad Test Diets, Harlan Sprague Dawley, Inc., WI 53713.

3DL-methionine, United States Biochemical Corp., Cleveland, OR.

4 Dextrose, Corn Products Company International, Englewood Cliffs, NJ.

5 Corn starch, United States Biochemical Corp., Cleveland, OH.

6 Mazola, Best Foods, Englebrook Cliffs, NJ.

7Mineral mix, formula based on AIN mineral mixture 76, without sodium chloride and chromium potassium sulfate (Appendix A).

8AIN vitamin mixture 76A, United States Biochemical Corp., Cleveland, OR.

9sodium chloride, MCB Manufacturing Chemists, Inc., Cincinnati, OH.

10choline bitartrate, United States Biochemical Corp. Cleveland, OH.

CHAPTER IV

RESULTS

Experiment One

Growth Rates

The mean growth rates for the five dietary groups are shown in

Figure 1. The low Cr/1.2% phytate group, had a significantly lower

(p < 0.0001) mean weight compared to the low Cr, low Cr plus 0.8%

phytate, and adequate Cr groups at week 13 of the study. The low Cr

group had a significantly lower (p < 0.05) end weight compared to the

adequate Cr/brewers' yeast group.

Dietary Analysis

Table 3 summarizes the phytate and chromium contents of the

various diets. The low Cr or basal diet was analyzed at 52 ppb

chromium, the low Cr/0.8% phytate diet contained 52 ppb chromium, and

the low Cr/1.2% phytate diet contained 22 ppb chromium. By analysis

the adequate Cr/Torula yeast diet contained 1.43 ppm chromium while

the adequate Cr/brewers' yeast diet contained only 39 ppb chromium.

Chromium values are based on analysis of samples on a flameless

atomic absorption spectrophotometer. The percent disodium salt of

phytic acid (c6

H6

Na12

o24

P6

) added to the diet was based on the

calculation of 58% phytate in the compound.

Glucose and Insulin

Mean plasma glucose removal rates (GRR) for the five groups are

presented in Table 4. There were no significant differences between

33

I .f ~

34

groups based on GRR at week 5 or 14. At week 8 the low Cr/1.2%

phytate group had a significantly higher (p < 0.05) GRR compared to

the other four groups. Only the low Cr and adequate Cr/brewers'

yeast groups showed a significant decrease (p < 0.05) in GRR from

week 5 to week 14. Insulin values, shown on Table 5, indicated a

significantly lower level (p < 0.05) in the low Cr/1.2% phytate group

as compared with all other groups.

Experiment Two

Growth Rates

Mean growth rates for the five groups are shown in Figure 2.

There were no significant differences between groups based on mean.

body weights. Growth rates of animals from Experiment One are not

comparable to those from Experiment Two since the former were male

and the latter female rats.

Dietary Analysis

The low Cr groups contained from 36 ppb to 49 ppb chromium as

indicated in Table 6. By analysis with flameless atomic absorption

spectoscropy, the adequate chromium group contained 1.62 ppm and the

adequate Cr plus Zn and Cu group contained 1.91 ppm chromium.

Tissue Analysis

Mean chromium concentrations in the liver are also expressed in

Table 6. There were no significant differences between groups based

on liver chromium concentrations.

35

Glucose and Insulin

Mean plasma GRR for the five groups are outlined in Table 7.

There were no significant differences between groups on either the

first or second glucose tolerance test. There was a significant

decrease (p < 0.05) in GRR with time only in the low Cr plus Zn and

Cu group. Insulin and glucose:insulin ratios, measured from plasma

collected 10 minutes after a glucose load showed no significant

differences (Table 8).

Experiment Three

Growth Rates

Figure 3 represents the mean growth rates for the four dietary

groups. There were no significant differences between mean body

weights of the four groups at the beginning or the end of the study.

The mean weights of the animals on this experiment were comparable to

the mean weights of the animals on Experiment One which were also

males.

Dietary Analysis

Table 9 summarizes the phytate and chromium content of the four 1 ·I . diet groups. The low Cr groups were analyzed as 62 and 37 ppb

chromium while the adequate chromium groups were analyzed as 1.74 and

2.29 ppm chromium. Total phytate by analysis in the low Cr/1.0%

phytate diet was 1.21% and 1.22% in the adequate Cr/1.0% phytate diet.

36

Glucose and Insulin

Mean plasma glucose values from the oral glucose tolerance test

(OGTT) are shown in Table 10. There were no significant differences

between groups at the fasting, 30 or 60 minute times; however, there

was a trend (p < 0.06) toward an effect of phytate at the 30 minute

time with the low Cr/1.0% phytate group having the highest glucose

value (171 mg/dl) and the adequate Cr group the lowest glucose value

(153 mg/dl). At the 120 minute time there was a significant effect

(p < 0.0001) of a Cr-phytate interaction and an effect (p < 0.01) of

phytate. The low Cr/1.0% phytate diet had the lowest glucose value

(98 mg/dl) at the one hundred and twenty minute time.

Table 11 shows the glucose increment which is based on the value

at each point on the glucose curve minus the fasting glucose value

(mg/dl). There was a significant effect (p < 0.04) of phytate at the

30 minute increment, but no effect was seen at the 60 or 120 minute

time period.

Insulin values from the OGTT are presented in Table 12. At

fasting, there was a significant effect (p < 0.004) due to.phytate

and the interaction of phytate and chromium (p < 0.05). There was no

significant difference between groups for the 30 minute insulin

values. The 60 minute values indicated a significant effect

(p < 0.002) due to chromium with the adequate Cr group without

phytate having the highest insulin level (12.9 ~U/ml) and the low

Cr/1.0% phytate group the lowest level (8.6 ~U/ml). At 120 minutes

there was a significant effect (p < 0.02) due to phytate and a trend

{p < 0.06) toward an effect of chromium. The adequate Cr group had

37

the highest level of insulin (8.8 ~U/ml) and the low Cr/1.0% phytate

group the lowest level of insulin (5.1 ~U/rnl).

400

350

300

250 ...-

O'l ---+.J ....1:: Oi 200 .,... <l)

::s: ~

'"0 0 co

150

100

50

0

-----LO¥J Cr /Tor yst Low Cr/Tor yst + 0.8% phy Low Cr/Tor yst + 1.2% phy

.. .. .. ... ..

•' •' ..

38

•'

.. ······· ..

/

/ ,/ Adeq Cr/Tor yst

Adeq Cr/brws' yst ...... ,....... . .,' •...•............... . . ,..· / . . ., . / .,. ., .,

••• / flllll',. . / /,. /

/ ,/ / /~

.. ·· /. / .. · . ~

... ····· /./ ,~'"' . "' .· / / .· / '/ .· /"

• ,/ '7

. / "' .· ./ ," ~""' .·· / / ~' . . / , .. · ./ / ---... I / ,. .,-:' / / .,.,

.... /,/ / .. /. '/ // : /q

.... ;; / .. · 11 ,""/

.... i., ..,-" .· i'l ,

.. ·· i I / . •/ // .··• .~I .· .; / . .; / . / .. / /

: I / ,• I ~

: I , :' I ,.,

/I /" •• I

•· I / . "/ -~

'Z~ ;' 7/

~~

1 2 3 4 5 6 7 8 9 10 11 1 2 13

Time 1n ~leeks

Figure 1. Mean body weight for groups during Experiment One.

39

TABLE 3

PHYTATE AND CHROMIUM CONTENT OF DIETS FOR EXPERIMENT ONE

1 % Phytate containing

compound added to diet

Phytate from analysis

% of diet

")

Chromiurn"-

Diet

Low Cr/Torula yeast

Low Cr/Torula yeast plus 0.8% phytate

Low Cr/Torula yeast plus 1.2% phytate

3 Adequate Cr/Torula yeast

Adequate Cr/brewers' yeast

0.00

1.38

2.07

0.00

0.00

0.29

1.00

1.44

0.29

0.29

content ppb

52

52

22

1430

39

1The disodium salt of phytic acid; 58% phytate by calculation.

2chromium analysis on Perkin-Elmer 5000 Atomic-Absorption­Spectrophotometer with Graphite Furnace and Zeeman background Correction (Appendix B).

3 Two parts per million chromium added to the diet as chromic chloride.

TABLE 4

PLASMA GLUCOSE REMOVAL RATES 1 FOR EXPERIMENT ONE

Group

Low Cr/Torula yeast

Low Cr/Torula yeast plus 0.8% phytate

Low Cr/Torula yeast plus 1.2% phytate

Adequate Cr/Torula yeast

Adequate Cr/brewers' yeast

(5)

(5)

(4)

(5)

(3)

1st GTT \~eek 5

5.4±1.2x

5.1±0.4

7.0±1.5

5.0±0.9

4.4±0.3x

N

(8)

(10)

(8)

(6)

(10)

2nd GTT Week 8

3.2±0.4ay

3.9±0.5a

6.4±1.lb

3.6±0.6a

3.3±0.2ay

N

(10)

(8)

(7)

(10)

(9)

1 Glucose removal rate expressed as mg/dl/min. + S.E.M.

2N is equal to sample size.

40

3rd GTT Week 14

2.6±0.2y

3.5±0.2

4.0±0.6

3.1±0.3

3.0±0.3y

abMeans with different superscript letters in the same column are significantly different (p < 0.05).

xyMeans with different superscript letters in the same row are significantly different (p < 0.05).

I f

TABLE 5

INSULIN VALUES FOR EXPERIMENT ONE

Group

Low Cr/Torula yeast

Low Cr/Torula yeast plus 0.8i, phytate

Low Cr/Toru1a yeast plus 1.2% phytate

Adequate Cr/Torula yeast

Adequate Cr/brewers' yeast

1N . 1 1 . 1s equa to samp e s1ze.

2rnsulin expressed as mean ± S.E.M.

(10)

(10)

(5)

(10)

(9)

41

Insulin2

~U/ml

32.8±2.1a

30.3±4.Ra

+ b 12.9_1.7

27.9±4.2a

36.2±5.8a

abMeans with different superscript letters in the same column are significantly different (p < 0.05).

I f l 1

300

250

200

.,.... 150 (])

3

100

50

..

0

.. .. · ..

, . .. ······ .

1 2

Low Cr -·-·-·-·-- Low Cr + Zn & Cu ··················· Low Cr + Zn --------- Adeq Cr + Zn ----- Adeq Cr

3 4 5

Time 1n Weeks

6 7 8

Figure 2. Mean body weight for groups during Experiment Two.

42

43

TABLE 6

CHROMIUM CONTENT OF DIET AND LIVER FOR EXPERIMENT TWO

Chromium content1

of diet Ch

. 1,2 ronnum content

Diet

Low Cr

Low Cr plus Zn and Cu

Low Cr plus Zn

3 Adequate Cr plus Zn and Cu

Adequate cr3

ppb

44

49

36

6010

7420

of liYer N ppb

(6)

(6)

(5)

(6)

(6)

17.55:tl. 7

17.89:t2.87

18.42:t2.19

19.06:t2.85

16.19:tl.34

1chromium analysis on Perkin-Elmer 5000 Atomic-Absorption­Spectrophotometer with Graphite Furnace and Zeeman background Correction (Appendix B).

2chromium content of liver expressed as means :t S.E.M.

3Five parts per million chromium added to the diet as chromium chloride.

TABLE 7

PLASMA GLUCOSE REMOVAL RATESl FOR EXPERIHENT TWO

Group

Low Cr

Low Cr plus Zn and Cu

Low Cr plus Zn

Adequate Cr plus Zn and Cu

Adequate Cr

(6)

(6)

(5)

(6)

(6)

1st GTT Week 4

3.5±0.3

3.7±0.3x

2.7±0.5

4.1±0.4

3.8±0.4

N

(5)

(5)

(4)

(6)

(6)

1 Glucose removal rate expressed as rng/dl/rnin. + S.E.M.

2N is equal to sample size.

44

2nd GTT \-leek 8

3.9±0.6

2.4±0.3y

2.3±0.2

2.9±0.4

3.4±0.5

xyMeans with different superscript letters in the same row are significantly different (p < 0.05).

)

TARLE 8

GLUCOSE AND INSULIN VALUES1

FOR EXPERIMENT T\.JO

Group

Low Cr

Low Cr plus Zn and Cu

Low Cr plus Zn

Adequate Cr plus Zn and Cu

Adequate Cr

(6)

(6)

(5)

(6)

(6)

Insulin lJU/ml

87.0±22.1

107.5±36.9

117.0±31.2

117.7±17.3

82.0±19.4

45

Glucose: insulin ratio

3.6±0.8

3.5±0.8

2.6±0.6

2.2±0.4

3.3±0.8

1 Values for insulin and glucose:insulin ratio are expressed as means ± S.E.M. Values are not significantly different.

2N is equal to the sample size.

+.J -'=

300

250

200

.~ 150 Q)

3

>, ""0 0

a:l

100

50

0

---············ ·······--------

Low Cr Low Cr + 1.0% phy Adeq Cr Adeq Cr + 1.0% phy

,

.. ········ __,. .. ·· _,., ... ··· .,.. .... / .. - / ... ·· ~~/ .·· ,~/

,"'/ ,, ,~ / .,, / .,., /

/'/ //

// //'

.·· /'/ .... // .... // .. ·· / ... ·· ~/ .. ~~ .·· ~ .. ··· // .· / .. ·· ~'/ .· / .... -'/ .· // .·· ~/ .. · // .· // .. · ~ .... <!'~ .· ~/

.··"""' ~~ ·""' .............

~

1 2 3 4 5 6 7 8

Time 1n Weeks

46

Figure 3. Mean body weight for groups during Experiment Three.

47

TABLE 9

PHYTATE AND CHROMIUM CONTENT OF DIETS FOR EXPERIMENT THREE

% Phytate containing Phvtate

compound "from analysis Diet added to diet % of diet

Chromium1

content ppb

Low Cr 0.00 0.23 62

Low Cr 1.0% phytate 1.72 1.21 37

')

Adequate Cr._ 1.0% phytate 1.72 1.22 1737

Adequate Cr 0.00 0.22 2287

1chromium analysis on Perkin-Elmer 5000 Atomic-Absorption­Spectrophotometer with Graphite Furnace and Zeeman background Correction (Appendix B).

2 Two parts per million chromium added to the diet as chromium chloride.

TATILE 10

GLUCOSE VALUES1

' 2 FROM OGTT FOR EXPERIHEt-!T THREE

Diet

Low Cr

Low Cr/1.0% phytate

Adequate Cr/1.0% phytate

Adequate Cr

Fasting 30 Min

90±3.6 159±6.0

80±3.0 171±4.9

83±2.3 160±6.0

82±2.4 153±3.5

1 Glucose expressed as mg/dl ± S.E.M.

2 There were 12 samples per group.

60 min

159±3.5

160±5.3

151±7.0

156±6.0

48

120 min

a 120±3.8

b 98~3.0

b 104±2.8

b 104±2.3

abMeans with different superscript letters in the same column are significantly different (p < 0.05).

TABLE 11

GLUCOSE INCREMENT1

'2

FROM OGTT FOR EXPERIMENT THREE

Diet

Low Cr

Low Cr/1.0% phytate

Adequate Cr/1.0% phytate

Adequate Cr

30 Min

69±6.7a

90±7.3b

77:t8.0ab

71::4.8ab

60 min 120 min

68:t5.0 22:t4.6

80:t6.8 17±3.8

67::8.2 2l:t3. 7

74:t6.9 21±3.7

49

1Glucose increment = fasting value - 30, 60, or 120 min value ± S.E.M.

2 There were 12 samples per group.

abMeans with different superscript letters in the same column are significantly different (p < 0.05).

TABLE 12

INSULIN VALUES 1 FROM OGTT FOR EXPERIMENT THREE

Diet

Low Cr

Low Cr/1.0% phytate

Adequate Cr/1.0% phytate

Adequate Cr

Fasting

5.8±l.la

5.1±0.7a

4.3±0.7a

8.3±0.Sb

30 Min

12.5±1.9

16.7±1.2

13.4±1.2

16.2±1.3

1Insulin values expressed as ~U/ml + S.E.M.

2 There were 12 samples per group.

60 min

9.3±0.8a

8.6±0.9a

11.0±1.2ab

12.9±0.8b

50

120 min

ab 6.8±0.7

b 5.1±0.8

+ ab 6.3_1.0

8.8±0.9a

abMeans with different superscript letters in the same column are significantly different (p < 0.05).

CHAPTER V

DISCUSSION

Growth Rates

The growth rates at week 5 of male rats fed the Torula yeast

based diet on Experiment One and Three were below the average for

male Sprague-Dawley rats (National Academy of Sciences-NRC, 1978).

The growth rate of the brewers' yeast group however was above the

average. The mean growth rates of animals on the Torula yeast based

diets were also below the growth rate of animals fed chow in this

lab. Female rats on Experiment Two, fed the same Torula yeast based

diet as those animals on Experiment One, were slightly above the

average for female Sprague-Dawley rats at week 5. Since the female

rat has a slower growth rate and smaller mature weight than the male,

the diet may not meet protein requirements for maximum growth of the

male rat. The unpalatability of the diet may also have contributed

to the poor growth rate. The low Cr/1.2% phytate group displayed an

extremely poor growth rate which may be due to a toxic effect of

sodium phytate. Animals in this group had a high mortality rate

(30%) compared to the other groups (0%) on the study. At autopsy the

kidneys of the low Cr/1.2~ phytate group appeared gray, mottled, and

enlarged. The kidneys of the rats in the 1.0% phytate groups on

Experiment Three had a similar but less severe mottled appearance.

Pathological microscopic examination was made of kidnev tissue from

animals in the adequate CR/1.0~ phytate group and the adequate Cr

group (without phytate) from Experiment Three. Calcium deposits and

severe nephrosis were f6und in the kidneys of the phytate group and

51

52

minimal or focal damage was seen in the kidneys of the non-phytate

group.

Diet and Tissue Analysis

All of the low Cr diets in Experiment One, Two and Three were

below 100 ppb chroMium, the level expected to produce a chromium

deficiency (Schroeder et al., 1963; Mertz, 1969). Samples of the

positive control diets tested showed some variation in concentration

from the amount of chromium added to the diet. The variation may be

due to uneven distribution of chromium in the mixing process and the

small quantity (0.150 g) used for analysis. However, the larger the

number of random samples tested the closer the mean was to the

expected value of 2.1 ppm for Experiment One and Three and 5.1 ppm for

Experiment Two.

Liver chromium concentration was measured in Experiment Two to

determine if chromium deficient animals would have a lower chromium

concentration, indicating the liver as a physiological pool for

chromium. The results, no significant difference between groups, do

not support or refute this hypothesis since there were no significant

differences between groups on the IVGTT. The liver chromium

concentrations from Experiment Two (16 to 19 ppb) were similar to

those (8 to 13 ppb) reported by Jain et al. (1981). The same studv

by Jain et al., (1981) also reported no significant difference

between groups for liver chromium concentration or glucose tolerance.

However, using a tracer technique in combination with chromium

analysis on the same study, Jain et al., (1981) calculated liver

chromium as 55~ exchangeable in the Cr sttpplemented group, 46~ in the

53

control group and 27% in the chromium deprived group. The results of

that study indicated the liver is a physiological pool for chromium,

but determination of absolute Cr levels and a radiotracer technique

together were needed to demonstrate those differences in the

physiological chromium pool.

The flameless atomic absorption spectrophotometer with Zeemen

background correction and a low temperature ashing method have

improved the sensitivity of chromium analysis over previous methods

used. Bovine liver powder, a standard reference material from the

National Bureau of Standards, was analyzed and 95% of the certified

value was recovered (84 ng/g out of 88 ± 12 ng/g).

Glucose and Insulin

Intravenous glucose tolerance tests (IVGTT) were performed

during Experiment One and Two to determine glucose removal rates

(GRR). In previous studies feeding a chromium deficient diet

resulted in a low GRR (Mertz, 1976). A removal rate below 2.5

mg/dl/min is considered a low or subnormal value while 4.0 to 4.5

mg/dl/min is considered normal or chromium adequate (Schroeder et

al., 1963; Mertz, 1969). Although the results presented in Tables 4

and 7 were not significantly different at any of the IVGTT, the low

Cr/Torula Yeast group on Experiment One, week 14, had a low GRR of

2.6, which may indicate inadequate chromium nutrition. Likewise in

Experiment Two the low Cr plus Zn and the low Cr plus Zn and Cu

groups had a low GRR. In general the low Cr groups in Experiment Two

had lower GRR than in Experiment One at week 8. The lower GRR on

Experiment Two may have been due to decreased chromium stores of

54

these rats since the dams had been maintained from weaning on a

chromium deficient diet. The dams kept on the Cr deficient diet

produced smaller and fewer pups per litter, as well as fewer litters

compared to dams on lab chow. Also the rats used in Experiment Two

were female and data are not available to compare the GRR of the male

and female rat.

The adequate Cr groups on Experiment One and Two did not have a

significantly higher GRR than the low Cr groups. The GRR were also

less than 4.0 mg/dl/min which indicated that the level of chromium in

the "adequate Cr" diet was not high enough to maintain normal glucose

tolerance. The form of chromium in the diet may have been bound or

alated during or before ingestion by the animals. Other studies have

demonstrated normal glucose tolerance by adding inorganic chromium to

the drinking water (2 to 5 ppm) (Schroeder, 1966; Jain et al., 1981);

however, previous studies in this lab produced low GRR with addition

of chromium to the water.

Brewers' yeast has been considered one of the best sources of

GTF chromium (Mertz et al., 1974); however, some brewers' yeast may

not be a good source of available chromium (Mertz, 1983) as evidenced

in Experiment One by the brewers' yeast group which demonstrated a

significant decrease in GRR from week 5 to week 14 with a final GRR

of 3.0 mg/dl/min.

The GRR of groups in Experiment One and Two tended to decrease

across time with the low Cr/Torula yeast, brewers' yeast and low Cr

plus Zn and Cu groups showing a significant difference (p < 0.05).

This effect has been partially attributed to an age-related decline of

55

islet secretory capacity (Reaven et al., 1983). This same studv

indicated that plasma insulin levels were lowest and insulin­

stimulated glucose utilization highest in calorie-restricted rats.

The same considerations may apply to the low Cr/1.2% phytate group

which had decreased food consumption, significantly lower insulin

levels, and significantly higher GRR compared to all other groups

during week 8 of Experiment One.

Mertz (1979) indicated that measurement of insulin values may be

a better indicator of differences in chromium status and possibly

more direct than measurement of glucose since the postulated role of

chromium is cofactor for insulin-receptor binding. Mertz (1979) also

indicated that chromium supplementation may correct either elevated

insulin levels or abnormal glucose tolerance or both.

On Experiment Three only the low Cr group at 120 minutes had

significantly higher glucose values. The fact that the highest

glucose increment was in the low Cr/1.0% phytate group may indicate

that chromium or some other trace element involved in insulin

metabolism was bound (unabsorbed). Insulin values in the low Cr

groups, though, were higher than the levels in the adequate Cr groups

instead of lower as expected. Statistically there was a greater

effect of phytate than of chromium or of the interaction between

phytate and chromium. A significant effect of phytate was seen at

the fasting and 120 minute insulin, at the 30 and 120 min glucose,

and on the 30 minute glucose increment. There was a significant

effect of the interaction of phytate and chromium at fasting insulin

and at the 120 minute glucose levels. A significant effect of

chromium on insulin levels was seen only at the 60 minute time

period.

56

Considering the toxicity effects observed in the kidneys of the

phytate groups there may be an indirect influence on insulin

production or secretion in the phytate group not measured by this

study. Since neither the glucose nor the insulin levels were

consistently higher in the low Cr groups, the chromium levels in the

adequate Cr groups may not have been high enough or bioavailable

enough to produce normal glucose tolerance.

CHAPTER VI

SUMMARY AND CONCLUSIONS

In these studies of the effects of phytate and zinc on the

chromium nutritional status of the growing rat, the following

observations were made:

1. The group fed a 1.2% phytate diet had significantly lower

weight gain and food consumption than all other groups.

2. A plasma glucose removal rate indicative of impairment of

glucose tolerance was produced in the low Cr group.

3. Plasma glucose removal rates in the adequate Cr groups were

not significantly different from the low Cr groups.

4. The groups fed low Cr diets with added phytate had higher

glucose removal rates than the control groups. Chromium

contamination from the sodium phytate did not account for

the differences in the glucose removal rates between the low

Cr phytate groups and the control groups.

5. Glucose removal rates were not significantly different

between groups fed a low Cr or adequate Cr diet with or

without added Zn and/or Cu.

o. Fasting, 30, and 60 minute glucose values from an OGTT did

not vary significantly with dietary group. The low Cr group

had a significantly higher glucose level at the 120 minute

time.

57

58

The following conclusions were drawn from the three studies:

1. Sodium phytate fed at a 1.0% or higher level in the diet may

produce a toxic effect on the kidney of the rat.

2. Zinc, fed at twice the recommended levels does not affect

chromium nutritional status as measured by glucose renoval

rates and liver chromium concentration.

3. Phytate exerted a greater effect on glucose and insulin

values from an OGTT than did chromium.

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APPENDICES

66

Step

Drying

Charring

Atomization

Burnout

APPENDIX A

PROGRAM SPECIFICATIONS USED WITH THE FLAMELESS

ATOMIC ABSORPTION SPECTROPHOTOMETER1- 3

Haximum temperature

oc

110

1000

26SO

2700

Ramp time sec

10

lS

0

1

Hold time sec

so

20

s

3

1 Sample volume of SO ~1 by manual pipet.

2Perkin Elmer Intensitron Hollow Cathode chromium lamp with 3S7.9 nm wavelength.

3 Argon gas.

67

68

APPE~DIX B

COMPOSITION OF MINERAL HIX

Ingredient ( of mineral mix

1 Calcium phosphate

Potassium citrate2

Potassium sulfate3

M . . d 4 agnes1um oxl e

5 Manganese carbonate

6 Ferrous sulphate

Zinc carbonate 7

P . . d 8 otasslum lO ate

Sodium selenite9

10 Vanadyl sulphate

C . b 11 upr1c car onate

Sucrose

1MCB, Manufacturing Chemists, Cinncinatti, OH.

2]. T. Baker Chemical Co. , Phillipsburg, N.J.

3J. T. Baker Chemical Co. , Phillipsburg, N.J.

4MCB, Manufacturing Chemists, Cinncinatti, OH.

5MCB, Manufacturing Chemists, Cinncinatti, OH.

6J. T. Baker Chemical Co., Phillipsburg, ~.J. 7

MCB, Manufacturing Chemists, Cinncinatti, OH.

8MCB, Manufacturing Chemists, Cinncinatti, OH.

50.00

22.00

5.20

2.40

0.35

0.48

0.16

0.29

0.13

0.01

0.03

18.95

69

a 'Sigma Chemical Company, St. Louis, MO.

10BDH Chemicals, Ltd., Poole England.

11MCB, Manufacturing Chemists, Cinncinatti, OH .

.. ··-~-·.·------------