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This is to certify that the
dissertation entitled
An Assessment of Monensin's Effect
Upon the Ruminant Small Intestine:
Na /K -ATPase Activity in Mucosal
Biopsies and Net Nutrient Absorption
presented by

Kristen A. Johnson

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Animal Science

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An Assessment of Monensin's Effect Upon the Ruminant Small Intestine:
Na*/K+-ATPase Activity in Mucosal Biopsies and Net Nutrient

Absorption

By
Kristen A. Johnson
A Dissertation
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy

Department of Animal Science

1987

ABSTRACT
An Assessment of Monensin's Effect Upon the Ruminant Small

Intestine: Na+/K+-ATPase Activity in Mucosal Biopsies and Net
Nutrient Absorption

By
Kristen A. Johnson

Studies were conducted to l) examine the effect of monensin upon
the Na+lK+-ATPase activity in bovine duodenal mucosal biopsies and
2) to examine the effect of monensin supplementation upon net nutrient
absorption. Two Angus heifers (233 kg) and two Simmental-crossbred
steers (309 kg) were fitted with duodenal cannulas approximately 10 cm
from the pylorus. Eighteen biopsies were removed per animal and were
examined for Na+/K+-ATPase activity, as measured by
ouabian-inhibition of 02 consumption in monensin concentrations of 0,
l. 5 and TD ppm. Total 02 consumption of the biopsies increased
(P<.Ol) from 5.59 nmol Ozlmg DM/min at 0 ppm to 8.18 nmol 02/mg
DM/min at l0 ppm. Ouabain-sensitive respiration increased (P<.Ol) with
increasing monensin concentrations from 2.76 nmol Ozlmg DM/min to 4.9
nmol Ozlmg DM/min at l0 ppm. Monensin addition to the diet of these
same animals (300 mg/hd/d) increased (P<.Ol) total 02 consumption
(20%) and ouabian-sensitive respiration (322). There was no effect
(P>.lo) upon NaTIKT-ATPase independent resipiration. In the second

study, four crossbred steers (363 kg) were catherized in the portal

potassium were altered by monensin supplementation. Magnesium
absorption weas increased (P<.02) with monensin supplementation (-.392
g/hr to .243 g/hr). These data indicate that monensin is having an
effect upon the small intestine, stimulating the Na+/K*-ATPase

pump. but that stimulation is not apparent in net nutrient transport
studies. It may be that the influx of sodium into the enterocyte is
stimulating the pump but not affecting other sodium dependent
processes. Altered magnesium absorption is best explained as a ruminal

effect with a stimulation of ruminal Na+lK+-ATPase.

Acknowledgements

There are many people who contributed in some way to this
dissertation and my graduate program and I wish to thank them.

I'd like to thank Dr. Mel Yokoyama, Dr. Duane Ullrey. Dr. Roy Emery,
Dr. Werner Bergen and Dr. Thomas Herdt for serving on the guidance
committee and for reviewing the manuscript.

I would especially like to thank Dr. Thomas Herdt for the time he
spent teaching me biopsy techniques, blood flow techniques and
especially for the enthusiasm he always had for my research. It was
always a pleasure to talk with him.

A special thanks to Dr. Kent Ames for all of his help and time
cannulating steers. He stuck with them even though it felt like we'd
cannulated an entire herd. Thanks also to Dr. Gerald Huntington, USDA,
Beltsville. for his technical advice. Other faculty members I'd like
to acknowledge for their time and advice include Dr. Elwyn Miller. Dr.
Steve Bursian and Dr. John Gill. .

To the people at the BCRC without whom research would be difficult
my sincerest thanks. Bud Peake. Dean Fischer, Bruce Truesdell, Les
Moore, Elaine Fink and all of the undergraduate help made everything
easier and were always willing to help.

Thank you also to Dr. Pao Ku, Phyllis Hhetter and Elizabeth Rimpau
for allowing me to use their laboratories and for teaching me to use
some of their euqipment. Special thanks to Jim Leisman for his help in
Statistical analysis of these data and for all of his stimulating
conversation. To Marilyn Emery, thank you very much not only for
facilitating finding and loaning of research material but for your

friendship and interest.

I would also like to graterlly acknowledge Judy Lentz fbr her
typing expertise and thank her for typing this manuscript. Thanks also
to Barb Sweeney fer her help in typing slides and data tables.

I would also like to sincerely thank Patty Dickerson and Trudy
Hughes fbr their help in sample collection. Pe0ple like those two make
graduate school bearable.

Finally, I would like to thank my family for their help and

support during my active graduate program.

I. INTRODUCTION. 0 O O O O O O O 0
II. REVIEN OF LITERATURE. . . . . .

A.
B.

c.

mfiirfio
.

H.

TABLE OF CONTENTS

Maintenance. . . . . . . .
Na+/K+-ATPase. . . . . . .
l. Enzyme Characteristics.
2. Na+/K+-A1Pase Activity.
3. NaTIKT-ATPase Activity i
Mineral Absorption . . .
. Calcium . . . . . .
. Phosphorus. . . . .
. Magnesium. . . . .
Potassium . . .

Ru i
Absorption of Sodium and Ch
h
t

0.00..0¢DO‘D..000:000.0

00.0.3.0...

Transcellular Sodium and C

Small Intestine
. Copper. . . . . .
. Zinc. . . . . . .
Selenium. . . . .
Lipid Absorption . .
Glucose Absorption .
Amino Acid and Peptide Absorp i n.
Monensin . . . . . . . . . . . . .

tom“ @m-wa"
0 0

0.000.110.0003..0..

Ill
or
or
O

l. Metabolism of Monensin. . . . . . . .
2. Monensin's Effect on the Na+/K+-ATPase.
Bl00d Flow Technique . . . . . . . . . . .

0-D.

(‘0
U

000000.0m000000:00000

O
0.000Q0000.

to
0000030000.

0"?

.0.00..0>'I'|.0.00

111. IN VITRO MEASUREMENTS 0F Ha*/K+-ATPase ACTIVITY
IntrOGUCti on O O O I O O O I O O O O O O 0
Materials and Methods. . . . . . . . . . .

A.
B.

1. Biopsy Technique. . . . . . . .
2. Handling of Biopsies. . . . . .
3. Oxygen Consumption Measurements
4. Calculations. . . . . . . . . .
Preliminary Experiments. . . . . .
Study l:
Biopsies. . . . . . . . . .

l. Animals . . . . . . . . . . .
2. Diets . . . . . . . . . . . .
3. Sampling. . . . . . . . . . .
4. Media . . . . . . . . . . . .
5. Analysis. . . . . . . . .

Study 2: The Effect of Monensin
Mucosal NaT/K+-ATPase Activity.
l. Animals . . . . . . . . . . .
2. Design. . . . . . . . . . . .
3. Diets . . . . . . . .
4. Sampling ..... . .
5. Analysis. . . . . . .

00.000M000.0
C

In Vitro Addition of Monen

s
p

00....fl.000.
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D
0.0.0.:00...0.0

C

00.00500...

0 - .
00-0..00.00Q000000.0-

0.3000000300000000

0.00Q00000

00.00.000.00.—l.000.000

0.00m00o00

m the Foresto
orption From

.00.0030000000.

do

00.0.000000fi3000’000.0'0

3
D
O

00000.:000000n0000.0.0

'U

0

.00000Q.00.0.m.0000000

£00.00.

0.0.0.0...0mn.0.00

d.

0.00.m0000-.

.0...0:0.00.0—I.000.000

In

.0000.-l.00000

F. ReSUItS. O O O O O C O O O O O O C O C O O O O 0-. O O

l. Study l:

2. Study 2:

In Vitro Addition of Monensin to Duodenal

Mucosal Biopsies . . . . . . . . . . . . . . . .

G. Discussion . . . . . . . . . . . . .

IV. IN VIVO:

NET NUTRIENT ABSORPTION

A. Introduction . . . . . . . .
B. Materials and Methods .....

\lOiUithd

9.
10.

1.

Animals . . . . . .

Di

Catheterization . .
Protocol. . . . . .

Bl

Sampling Handling .
Assays. . . . . . . . .

a.
b.
c.
d.
e.
f.

Mi
a.
b.
c.
d.
e.

Arterial and Portal Blood Concentratio

a.
b.
c.
d.

et. 0 O I O O I O O 0
00d Flow Measurements

L-Lactate . . . . .
Glucose . . . . . .
Blood Urea Nitrogen
Alpha Amino Nitrogen.
Total Lipid . . . . .
p-Aminohippuric Acid.
neral Analysis. . . . .
Chloride. . . . . . .
Phosphorus. . . . . .
Selenium. . . . . .

Magnesium, Calcium. Copper and Z n

Potassium and Sodium. . . . .
Statistical Analysis. . . . . . .
Calculations. . . . . . . . . . .
CO Resu1t50 O O O 0 O I O O O O O O O O O

The Effect of Monensin Supplementation on
Duodenal Mucosal Na+/K*-ATPase

ACtEVEty. 0 0 . 0

1'

=00.0fi000.000.00000.00000

Minerals. . . . . . . . . . . . . .
Lactate. Glucose and Total Lipid. .
Alpha Amino Nitrogen and Blood Urea
Blood Flow Estimates. . . . . . . . . . .
2. Portal Arterial and Net Absorption Differences.

O O *0 O O Q 0 O O O O 0 O O O O O O O O O O O O O I

e
e

000.0.000000000000000.00000

g n.

v. DISCUSSIO". O I O O O O O O O O O O O O O O O O O O O O 0

v1. APPENDIX. 0 O O O O O O O O O O O O O O O C O O O O O O O

VII. LITERATURE CITED.

00000100000.00000...0.000...0.0

lOS

LIST OF TABLES

Page

TABLE l. A Summary of Studies Involving Monensin as a Cellular
Perturbant O O O O O O O O O O O O O O O O O O O O O O 42

TABLE . Modified Krebs-Henseleit Buffer. . . . . . . . . . . . 52

2
TABLE 3. Composition of Heifer Ration . . . . . . . . . . . . . 57
4

TABLE . Composition of to Steers Ration. . . . . . . . . . . . 58
TABLE 5. Mean Mucosal OI Consumption, NaT/K+-ATPase
Dependent and ndependent Respiration of Duodenal

Mucosa with Different Concentration of Monensin Added
to the Media 0 O O O O O O O 0 O O O O O O O O O O O O 62

TABLE 6. Mean Mucosal 0 Consumption, Na+lK+-ATPase
Dependent and Independent Respiration of Duodenal
Biopsies Taken From Control and Monensin Supplemented
Beef Cattle. . . . . . . . . . . . . . . . . . . . . . 64

TABLE 7. Diet Composition for Net Absorption Studies. . . . . . 70

TABLE 8. Mean Arterial and Portal Blood Concentrations of
Calcium and Phosphorus From Control and Monensin
SupplementedSteers..................81

TABLE 9. Mean Arterial and Portal Blood Concentrations of

Magnesium, Copper, Zinc and Selenium From Control and
Monensin Supplemented Steers . . . . . . . . . . . . . 82

TABLE 10. Mean Arterial and Portal Bl00d Concentrations of
Sodium. Potassium and Chloride From Control and
Monensin Supplemented Steers . . . . . . . . . . . . . 84

TABLE ll. Mean Arterial and Portal Blood Concentrations of
L-Lactate, Glucose and Total Lipids in Control and
Monensin Supplemented Steers . . . . . . . . . . . . . 85

TABLE l2. Mean Arterial and Portal Blood Concentrations of Alpha
Amino Nitrogen (AAN) and Blood Urea Nitrogen (BUN)
From Control and Monensin Supplemented Steers. . . . . 87

TABLE 13. Blood Flow Estimates (L/hr) From Control and Monensin
Supplemented Steers. . . . . . . . . . . . . . . . . . 88

TABLE 14. Mean Portal-Arterial (P-A) Differences and Net
Absorption Rates (NA) in Control and Monensin
Supplemented Steers: Calcium and Phosphorus . . . . . 89

TABLE l5. Mean Portal-Arterial (PA) Differences and Net
Absorption Rates (NA) in Control and Monensin

Supplemented Steers: Magnesium, Copper, Zinc and
Scleni um O O O O O O O O O O O O O O O O O O O O O O O 90

. Page

TABLE l6. Mean Portal-Arterial (P-A) Differences and Net
Absorption Rates (NA) in Control and Monensin
Supplemented Steers: Sodium. Potassium and
Ch10r1de O O O C C O O O O O I O O O O O O O O O O O O 92

TABLE 17. Mean Portal-Arterial (PA) Differences and Net
Absorption Rates (NA) in Control and Monensin
Supplemented Steers: Lactate, Glucose. and Total
Lipids . . . . . . . . . . . . . . . . . . . . . . . . 94

TABLE 18. Mean Portal-Arterial (PA) Differences and Net
Absorption (NA) in Control and Monensin Supplemented

Steers: Alpha Amino Nitrogen (AAN) and Blood Urea
Ni trogen (BUN) O O O O O O O O O O O O O O O O O O O O 95

TABLE l9. A Summary of Recent Data Concerning Monensin Addition
to Beef Cattle Diets and Its Effect on Net Nutrient
Absorption O I O O O O O O O O O O O I O O O O O O O .104

LIST OF FIGURES

Page
FIGURE l. Model for Electrogenic Sodium Transport . . . . . . . . . .20

FIGURE 2. Coupled Absorption of Sodium and Organic Salutes. . . . . .21
FIGURE 3. Models for Chloride Absorption. . . . . . . . . . . . . . .23

FIGURE 4. Schematic Views of the Structure of Monensin and Monensin
SOdi um. O O O O O O I O O I O O O O O O O O O I O O O I O .41

FIGURE 5. A Schematic View of the Mechanism of Monensin's Action in
the ca] 1 Membra ne 0 O O O O O O O O O O O O O O O O O O O O 44

FIGURE Al. Inhibition by Ouabain of Respiration in duodenal mucosa . lO4

Introduction

A growing animal has two types of energy needs. “maintenance" and
”gain“. Absorbed nutrients are first partitioned to maintenance
functions and then to gain. Estimates of the feed energy used by a
feedlot steer or heifer for maintenance have ranged from 40 to 63% (Van
Es, 1972; Johnson. 1984). This wide fluctuation in maintenance
requirements is a serious concern to an industry whose goals include
maximum production (e.g.. gain) with minimal input (e.g., feed costs).
Our goals as animal scientists are: 1) to understand maintenance
functions and requirements; and 2) to manipulate and minimize
maintenance costs. thereby increasing the feed energy available for
gain.

One method of increasing animal efficiency is ionophore
supplementation of feedlot rations. RumensinTM. monensin sodium.
is the ionophore used most commonly. Goodrich et al. (1984) examined
and summarized data on 16,000 head of cattle and found that monensin
supplementation decreased intake 6.4%, increased rate of gain 1.61 and
decreased feed/TOO kg of gain 7.5%. There was no effect on carcass
composition.

The mode of action of these compounds has received considerable
attention. In a review article. Schelling (1984) identified seven
proposed modes of action for monensin. These modes of action included:
1) modified acid [propionate] production; 2) modified feed intake; 3)
change in gas pr0duction; 4) modified digestibilities; 5) changes in

protein utilization; 6) modified rumen fill and rate of passage; and 7)

2
other ruminal modes of action. .Modifications in these parameters are
insufficient to account for the magnitude of the feed efficiency
response seen with monensin supplementation (Bergen and Bates. 1984).
This suggests that ionophores have effects beyond those observed in the
rumen.

Data reported by Davison (1984) indicate that ionophores pass
through the rumen unchanged. This might expose the epithelial cells of
the intestine to concentrations sufficient to alter their physiological
state. Ionophores function by facilitating ion transport (Pressman,
1976). Monensin and lasalocid (another widely used ionophore in beef
cattle diets) have affinities for sodium and potassium ions.
respectively. This has led to speculation that these ionophores might
disrupt the normal sodium and potassium gradients seen across the
mucosal cell (Bergen and Bates, 1984). 7

The balance of sodium and potassium ion concentrations across the
intestinal cell is extremely important in nutrient transport processes.
Many of the amino acids and all of the glucose absorbed from the
intestinal lumen are transported by sodium dependent processes (Munck,
1981). A concentration gradient of high luminal sodium (l4O mEq) and
low mucosal sodium (l4mEq) is maintained by the Na/K+-ATPase pump.

This pump exchanges three sodium ions for two potassium ions. with the
hydrolysis of one ATP. Glucose and some amino acids flow with the
sodium ions across the mucosa. An ionophore within the epithelial cell
membrane would facilitate sodium influx, and might stimulate amino acid
or glucose absorption and igcreasing the Na+/K+-ATPase pump

activity. Alteration of potassium flux across the cell membrane would
affect the mucosal cell and thereby alter nonsodium-linked amino acid

transport (Bergen and Bates, 1984).

3

The effects of ionophores on the intestinal mucosa have not been
investigated. Since the ruminal effects are not sufficient to account
for the increased efficiency seen with ionophore addition to ruminant
diets and since ionophores have been shown to affect glucose transport
across other types of cells (Austic and Smith. 1980, Nordenberg et al.,
1984) the following studies were conducted to assess the possible
alteration of nutrient absorption and gastrointestinal maintenance

costs with ionophore addition.

Review of the Literature
Maintenance

An animal's maintenance requirement is defined as the amount of daily
dietary intake which will result in neither gain nor loss of body
energy (Johnson, 1984). Maintenance costs may be broken down into
three areas: environmental factors; animal to animal variations and
physiological or compositional changes. Environmental factors include
the climate or season. A decline in temperature below the
thermoneutral zone may increase maintenance requirements 40-50%
(Johnson, 1984). Other environmental factors include: level of
alimentation, diet and activity. These may alter maintenance costs
10-30% (Johnson, 1984). Animal to animal variation, including
differences in genetics, sex, sweat glands and hair color, may alter
the amount of energy partitioned to maintenance by 10 to 30% (Johnson,
1984). Physiological or compositional changes such as lactation, age,
vital organ mass and muscle mass may, under different circumstances,
contribute a 5 to 15% change in maintenance costs. The greatest
changes occur when alterations in protein turnover or ion pumping take
place. These physiological factors may alter maintenance costs
20-30%. I

Milligan (1971) has suggested that ion pumping contributes
substantially to maintenance costs. The maintenance of sodiun and
potassium gradients across the cell plasma membrane by
Na+/K+-ATPase contributes 30-40% of the total energy expenditure of
the brain, liver and skeletal muscle (Ismail-Beigi and Edelman, 1971).

Other work done in Milligan's group (Gregg and Milligan, 1980a,b; Gregg

5
and Milligan, 1982a,b; McBride and Milligan. 1984; McBride and
Milligan, 1985) confirms this large contribution of Na+/K*-ATPase.
Muscle biopsies from calves and lambs of different ages show that young
animals have a much greater total oxygen uptake indicating a greater
Na+/K+-ATPase activity and greater energy costs. Similar results
have been shown in hepatocytes (McBride and Milligan, 1985). Data in
muscle, liver and duodenal biopsies indicate that lactation also
increases the aerobic energy consumed and energy requirements of the
tissue involved (Milligan and McBride, 1985).

Cold exposure (Gregg and Milligan, 1982a), level of energy intake
(McBride and Milligan, 1985). and age of the animal alter energy costs.
This effect of physiological state of the animal alters maintenance
costs (Milligan and McBride, 1985).

Na+/K*-ATPase
Enzyme Characteristics ‘

Experiments with various tissues, frog skin, nerves, muscle, red
blood cells and enterocytes, have shown that sodium is transported from
the cytosol to interstitial fluid against an electrochemical gradient
(Skou, 1965). This active transport of sodium'was shown to be ATP-
dependent (Hoffman, 1960). dependent on extracellular potassium
concentrations (Taylor, 1962). inhibited by cardiac glycosides (Skou,
1965) and showed a coupling between sodium efflux and potassium influx
(Taylor, 1962). These and other sets of data lead to the proposal of a

transport system for sodium that fulfilled the following criteria
(Skou, 1965):

1) located in the cell membrane
2) have greater affinity for Na+ than K+ inside the cell
membrane
3) have greater affinity for K+ than Na+ outside the cell
membrane
4) be able to catalyze the hydrolysis of ATP and convert the
energy to a transport process
5) be able to hydrolyze ATP at a rate dependent on internal
Na+ concentration and external K+ concentration
6) be found in all cells which have active Na+/K+ linked
active transport
7) have a close correlation between the effect of cardiac
glycosides and cation transport
A Na+/K+ATP—hydrolyzing enzyme system was identified and
characterized by Skou (1957, 1960, 1961, 1963, 1964) in nerves, brain,
and kidneys. This'enzyme requires ATP, Na+, K+ and MgT. Further
work identified a similar enzyme system in intestine (Taylor, 1962),
red blood cells, liver, muscle and many other tissues. The differences
in the enzyme ATPases isolated from the various tissues include
differential sensitivity to cardiac glycoside concentrations, different
activities at different pH's and differential sensitivity to Mg+
concentrations (Skou, 1965).
The stoichiometry of the NaT-K+ exchange was determined to be
2-3 Na+ ions for 2 K+ ions (Sen and Post, 1961). In several
studies with phospholipase A and C, additives of detergents (DOC and

lauryl sulfate) and additions of fatty acids (oleic and stearic acids)

7
it became obvious that the enzyme system required a stable, organized
lipOprotein structure (Glynn, 1962; Skou, 1962).

The enzyme has also been shown to be inhibited by high
concentrations of oligomycin (Van Groningen and Slater; 1962; Jobsis
and Vreman, 1963), sulfhydryl inhibitors (Skou, 1963) and cardiac
glycosides (Skou, 1963). It is unaffected by 2,4-dinitr0phenol,
aldosterone and insulin (Bonting et al., 1961). More recent evidence
indicates that insulin stimulates the NaT/K+-ATPase in skeletal
muscle (Moore, 1983).

The enzyme is composed of two noncovalently bound subunits (Harris
and Stahl, 1984). These are an a-subunit that contains the catalytic
site and a 6-subunit. Ouabain acts to modify the a—subunit's
confbrmation (Anner, 1985) and thereby inhibits enzyme function. The
proposed mechanism of enzyme action of the ATPase involves protonation
of an NHZ group on the a-subunit (Jorgensen et al., 1982). At this
time the enzyme is in the E2 fbrm which has a high affinity fbr K+
and a low affinity fbr Na+ and ATP. Deprotonation or the E1 fbrm
has a high affinity fbr Na+ and ATP but low affinity fbr K+. This
protonation-deprotonation reaction may cause a change in tertiary or
quarternary structure causing a "flip-flop" of the a-subunit from a
hydrophilic to hydrophobic environment. The suggested fonction of the
B-subunit is to orient the a-subunit (Skou, 1982).

Na+/k+-A1Pase Activity

In the intestine Na+/K+-ATPase has been identified in the

basolateral membrane of the intestinal cell (Fugita et al., 1971;

Parkinson et al., 1972). It has also been shown to be associated with

8
intestinal electrolyte transport, glucose transport in rat ileum, and
maintenance of an electrochemical gradient across the enterocyte
(Charney et al., 1975).

Data presented by Quigley and Gotterer (1968) indicate that the
Na*/K+-ATPase in rat intestinal mucosa is associated with, but not
stimulated by amino acid addition. Increased sodium and potassium
concentrations did stimulate ATPase activity 8 fold over control
concentrations. More recent data also indicate that increased
intracellular sodium concentrations stimulate the activity of the
Na*/K+—ATPase (Marunaka, 1986). Other stimulation of the pump was
seen by Charney et a1. (1975) when adrenal steroids, methylprednisolone
acetate and deoxycorticosterone acetate injections were given to rats.
Jejunal, ileal and colon segments were examined fbr ATPase activity.
Their data indicate that where gluco and mineralocorticoid treatments
increased water and electrolyte transport, the Na+/K+-ATPase also
increased in activity. This is unlikely to be a direct effect of
adrenal steroids on the pump but may be secondary to an enhancement of
sodium absorption. Silva et a1. (1975) examined the effect of
aldosterone and adrenalectomy upon the colon mucosa. Adrenalectomy
reduced Na+/K+-ATPase activity 50%, and replacement of aldosterone
increased activity although not quite to control levels.

Another finding of this group (Silva et al., 1975) was a
progressive decrease in Na+/K+-ATPase activity from the duodenum to
the colon. In rats, duodenal Na+/K+-A1Pase activity was 14.69 umol
Pi/mg protein/hr, jejunal 11.35 umol Pi/mg protein/hr, ileal 7.72 umol

Pi/mg/protein/hr and colon 4.48 umol Pi/mg protein/hr (Pi=inorganic

9

phosphate). This decreasing gradient of activity is in agreement with
data of Charney et al. (1974) and later data of Murray and Wild
(1980).

Fasting has been shown to decrease Na+/K+-ATPase activity in
rat jejunum (Murray and Wild, 1979) and sheep duodenum (McBride, 1984).
This would seem to indicate that the enzyme may be inducible. Data
presented by Schiffl and Loeschke (1977) indicate that adaptation
responses in the cecal mucosa to diet differences, are due to an
induction of more ATPase molecules per unit of basolateral cell
membrane.

In summary, the Na+/K+-ATPase couples the flow of cations
against their electrochemical gradients LNa+J and the flow of cation
along their gradients LK‘J to ATP hydrolysis, maintaining critical
sodium and potassium balances in the cell. The enzyme is located on
the basolateral membrane of the intestinal cell and has been shown to
react to changes in diet, diet composition and hormone concentrations
(Skou, 1982).

Na+/K+ Activity in Ruminant Duodenal Biopsies

The available data in ruminants on duodenal mucosal
Na+/K+-ATPase include two studies done by McBride (1984). Total
oxygen consumption, ouabain-sensitive and ouabain-insensitive
respiration rates were measured for the duodenal mucosa of nonlactating
and lactating cows (McBride and Milligan, 1984). In this study 55% of
the total mucosal respiration of cows at peak lactation was attributed
to Na‘lK*-ATPase, ouabain-sensitive respiration. In mid-lactation
and during the dry period the portion of oxygen consumption inhibited

by ouabain (Na+/K*-A1Pase) declined to 34-35%. Total oxygen

IO

consumption rates ranged from 6.49 ul OZ/mg DM/hr (pregnant,
nonlactating) to 8.20 ul Oz/mg DM/hr at 22-25 weeks into lactation.
The ouabain-sensitive portion of this oxygen consumption ranged from
2.45 ul Oz/mg DM/hr (pregnant, nonlactating) to 3.99 ul Ozlmg DM/hr
at 5 weeks of lactation. These data indicate that Na+/K+-ATPase
accounted for a major portion of duodenal maintenance costs, and that
these costs change with different physiological states.

In another study McBride (1984) examined the effect of digestible
energy intake (starvation, low and high energy) on the
Na+/K+-ATPase in sheep mucosal biopsies. In this study there was
no significant difference in total mucosal oxygen consumption but
ouabain-sensitive respiration was different (P<.Ol). During starvation
biopsies consumed 1.48 nmol Ozlmg DM/min. At low energy intake 2.69
nmol 02/mg DM/min were consumed and at high energy intakes 3.69 nmol
02/mg DM/min were consumed. These results indicate that the
Na+/K+-ATPase activity contributes significantly to the magnitude
of energy consumption by the mucosa and that this portion of energy is
variable with level of digestible energy intake.

Mineral Absorption

The major factors that influence mineral availability in the
digestive tract of ruminants are dry matter content, pH and location in
the tract (Cragle, 1973). The diet influences all of these factors.
The primary effect of pH is on mineral solubility. A decrease in pH
will allow a specific mineral to become more ionized allowing for
absorption across membranes (Georgievskii, 1981). This is particularly

true of calcium and magnesium.

11
Calcium

There is some disagreement as to where in the ruminant intestinal
tract calcium is absorbed. Chandler and Cragle (1962) described net
absorption of calcium from the abomasum. Other workers (Phillipson and
Storry, 1965; Liebholz, 1974) have also demonstrated calcium absorption
from the intestine. Absorption estimates vary from 10-50% depending on
the age of the animal being investigated and the diet fed (Leibholz,
1974; Georgievskii, 1981). Calcium absorption may be inhibited by
excess phosphates and magnesium ions and stimulated by vitamin D
(Georgievskii, 1981).

The mechanisn of calcium absorption by the small intestine has
been actively investigated. There are four proposed mechanisms of
intestinal calcium absorption (Nasserman, 1981). In one model the
calcium diffuses across the brush border and down a gradient as a free
ion (Rasnussen et al., 1979). It then complexes with binding proteins
(such as mitochondrial binding proteins) maintaining low intracellular
ion concentrations. Calcium extrusion is via an energy demanding
process across the basolateral membrane. The pumps on the basolateral
membrane are considered to be one of two types. A calcium stimulated
ATPase has been fbund on the brush border and basolateral membrane
(Melancon and DeLuca, 1970; Ghijsen et al., 1982). A second possible
mechanisn is a calcium-sodium exchange mechanism or a
sodium-calcium-dependent ATPase fbund on the basolateral membrane
(Birge et al., 1972, 1974; Martin and DeLuca, 1969).

The third proposed mechanisn involves endocytosis. Luminal
calcium is incorporated into membrane vesicles at the brush border

membrane (Nasserman, 1981) and moves into the cell cytosol where it

12
associates with lysosomes. Secondary lysosomes are formed, where
calcium binding would occur, and eventually calcium is released by
exocytosis. This mechanism involves mitochondrial accumulation of
calcium from the cytosol with subsequent transport to the basolateral
membrane (Nehringer et al., 1978).

Finally, a paracellular pathway has been proposed. This pathway
allows fbr calcium transport in either direction and is thought to be
the primary mechanism of calcium secretion into the intestine. The
flux of calcium would depend on the direction of solvent flow and the
electrochemical potential gradient (Nasserman, 1981).

la,25-dihydroxycholecalciferol (Vitamin 0) may be a factor in each
of these pr0posed mechanisms. Calcium-binding protein, thought to be
induced by vitamin D, is likely to be extremely important in absorption
of calcium from the intestine but may not be the only mechanisn.
Vitamin D has also been shown to increase the activity of
calcium-ATPase (Melancon and DeLuca, 1970; Ghijsen and Van Os, 1982)
and to modify the hydrocarbon side chains of the phospholipids present
in the brush border membrane (Goodman et al., 1972; Max et al., 1978).
Both of these actions would increase calcium absorption from the
intestine and would fit with one or more of the proposed models.

The absorbed calcium moves to the liver through the portal vein
where it fbrms new compounds and is released into the circulation
(Georgievskii, 1981). Feed and endogenous calcium is eliminated
primarily through the gastrointestinal tract, although negligible
amounts of calcium are excreted in the urine (Georgievskii, 1981).
Normal plasma calcium values for cattle range from 6.4 to 10.9 mg/dl

(Georgievskii, 1981).

13

The critical balance of calcium absorption and excretion is
mediated through parathyroid hormone. A decrease in plasna calcium
level stimulates an increased parathyroid hormone release which
stimulates increased lo,25 (0H)ZD3 production in the kidney. lu,25
(OH)ZD3 acts on the gut to produce calcium binding protein and
increases intestinal absorption (DeLuca, 1979).

Phosphorus

Phosphorus absorption in ruminants is thought to occur in the
small intestine (Bruce et al., 1966; Pfeffer et al., 1970; Grace et
al., 1974; Ben-Ghedalia et a1, 1975; Kay and Pfeffer, 1970), primarily
the duodenum. There is recent evidence of some ruminal absorption of
phosphorus through a Na+/K+-ATPase mechanisn (Breves et al., 1986)
but this is not thought to be as significant as intestinal absorption.

Phosphate, in the rat, is absorbed by active transport (Harrison
and Harrison, 1961), is stimulated by vitamin D and is dependent upon
sodium. The proposed model indicates uphill transport of phosphate
across the brush border energized by the downhill transport of sodium.
A common membrane component present in the brush border for phosphate
and sodium transport has been proposed (Nasserman, 1981). PhOSphate
then diffuses through the cytosol, without entering the cytoplasmic
pool of phOSphate, and diffbses through the basolateral membrane
(Peterlik and Hasserman, 1978). This process has also been
demonstrated to be saturable (Scott et al., 1984; Care et al., 1980).

Once absorbed inorganic phosphates undergo transfbrmation to
organic phOSphorus compounds (Georgievskii, 1981). Tissue phosphorus

incorporation is variable and dependent on the state of the tissue

I4
(i.e., growing, lactation). Normal plasma phosphorus values range from
3.5 to 7.8 mg/dl (Georgievskii, 1981).

The major route of phosphorus excretion is the digestive tract
(Care et al., 1980). Dietary phosphate and salivary phosphate are
available for absorption or reabsorption in the gastrointestinal tract
(Care et al., 1980). A second route of excretion of phosphate is the
urine (Georgievskii, 1981; Scott and Buchan, 1985). The extent to which
these routes are utilized is dependent upon the diet. Generally,
concentrate feeding increases urinary excretion of phosphorus and
decreases salivary secretion as compared to hay feeding (Scott and
Buchan, 1985). Whether this shift is a form of feed effect or a
digestibility effect or both is unclear. Earlier work by the same
authors indicates that digestibility of the diet may affect renal
response to phOSphorus (Scott et al., 1984).

Parathyroid hormone affects phosphate balance as well as calcium
balance. Phosphate elimination through the urine is increased with
increasing parathyroid hormone through decreased reabsorption (McIntosh
and Tomas, 1978). la,25(OH)ZD3 has also been suggested to affect
phosphate absorption in the intestine but it is not known if this is a
direct effect on phosphorus or a secondary effect of calcium absorption
(Care at al., 1980). Calcium is not essential fbr phosphorus
absorption (Hasserman, 1981) but does affect the amount of absorbed
phosphorus (Care et al., 1980). Excess calcium, magnesium or iron may
result in the formation of insoluble phosphate compounds while a
decrease in the Ca/P ratio below 0.3 will also decrease phosphate

absorption (Care et al., 1980).

15
Magnesium
Magnesium in blood is found either ionized (65%) or protein
(albumin) bound (35%) and is in dynamic equilibrium (Georgievskii,
1981). There is disagreement as to the site of magnesium absorption.
Until 1970 the concensus was that magnesium was absorbed by either
diffusion (ordinary or facilitated) or active transport in the duodenum
and upper large intestine (Martens and Rayssiquier, 1980). Since that
time evidence for absorption of magnesium from the rumen epithelium has
mounted. Ben-Ghedalia et a1. (1975) demonstrated that the fbrestomachs
(befbre the pylorus) absorbed magnesium. Tomas and Potter (1976)
further defined the site of absorption to be the rumen. With the
development of cannulation techniques, magnesium transport across
isolated rumen epithelium has been actively studied. Active transport
is the proposed mechanism (Martens and Rayssiquier, 1980) based upon
evidence of transport across a concentration gradient, dinitrOphenol
inhibition, saturation kinetics, temperature sensitivity and competive
inhibition. Additional evidence of inhibition of magnesium transport
exists when Na+/K+-ATPase inhibitors, such as ouabain, are added.
Care et a1 (1984) using a dorsal rumen pouch demonstrated: 1) net
magnesium absorption against an electrochemical gradient, 2)
sensitivity to K+/Na+ ratio (increased K+/Na+ leads to
decreased net efflux) and 3) an inverse relationship between calcium
concentrations in the rumen and net absorption rate of'magnesium.
Martens (1985a) further demonstrated that magnesium efflux was ATP
dependent supporting the conclusion that magnesium transport is an

active transport process, that it is not sensitive to changes in water

16
absorption or osmotic pressure (Martens, 1985b), and that magnesium
transport is a saturable process (Martens, 1983; Gabel and Martens,
1985).

Absorbed magnesium is deposited in bone and muscle tissue. The
major mechanisms for elimination of nonabsorbed and endogenous
magnesium is elimination from the gastrointestinal tract (Georgievskii,
1981). Urinary excretion of magnesium is small but does play a
significant role in magnesium homeostasis. Magnesium levels in the
plasna range from 1.8 to 2.7 mg/dl (Georgievskii, 1981).

Potassium

Normal serum potassium levels in growing cattle are 18-20 mg/dl
(Georgievskii, 1981). Potassium is absorbed by all segments of the
digestive tract, particularly the rumen, abomasum and duodenum. The
fraction of absorbed potassium that each of these areas account fbr is
diet dependent (Paquay, 1969). _

Absorption mechanisns for potassium within the rumen have been
investigated by Scott (1974). His findings indicate that potassium
absorption from the rumen is dependent on the concentration of
potassium present in the rumen fluid. In one of his studies he
infbsed sheep with various levels of potassium through rumen cannulas.
At the lowest level of potassium infbsed, rumen potassium levels
reached 40 mEq/l. The absorption rate at this concentration was 6
mEq/hr. At the highest level, rumen concentrations reached 110
meq/L and absorption rates were 20 mEq/hr. The mechanism by which
potassium is absorbed through the rumen wall is proposed to be via a

concentration gradient established by higher potassium in the rumen

fluid than in the blood. This concentration gradient is likely to

 

17
exceed the electrical potential gradient so that net movement of
potassium is to the blood stream (Scott, 1967).

There is also omasal absorption of potassium (Engelhardt and
Hauffe, 1975). Ten percent of the potassium entering the omasum was
absorbed. The mechanism by which the absorption occurs has been
suggested to be similar to that of the rumen epithelium (Engelhardt and
Hauffe, 1975).

The mechanism of potassium absorption from the duodenum, jejunum
and ileum are not elucidated far the ruminant animal but are well
investigated in the rabbit, rat, cat and dog. Rabbit ileum data
indicate that potassium transport across the mucosal membrane is a
passive process driven by an electrochemical gradient (Schultz et al.,
1974). This evidence further suggests that the mucosal membrane is
essentially impermeable to potassium and that intracellular potassium
levels are maintained by the pump at the basolateral membrane. All the
potassium moving across the epithelium appears to be maving
extracellularly through tight junctions (Schultz et al., 1974).

Potassium is eliminated primarily through the kidneys, with a
snall percentage eliminated in the feces (Ward, 1966). At the level of
the kidney, aldosterone and deoxycorticosterone are the mechanisns for
maintaining homeostasis. As the concentration of sodium in the plasma
decreases relative to potassium, aldosterone acts to decrease urinary
sodium and increase potassium excretion thus maintaining a Nat/K+
balance (Georgievskii; 1981).

Absorption of Sodium and Chloride from the Forestomach

Sodium and chloride are absorbed by all segments of the ruminant

forestomach (Edrise et al., 1986). These minerals are also actively

18
secreted into each of these compartments from the plasma or saliva.
The omasum is the part of the stomach that has the greatest absorption
rate of sodium and the greatest secretion of chloride ions. Engelhardt
and Hauffe (1975) estimated that 25% of the sodium entering the omasum
was absorbed. This is in agreement with Edrise et a1. (1986) who
estimated 40-60% of the sodium was absorbed. Data from studies
measuring chloride absorption across the rumen and omasal epithelium
(Engelhardt and Hauffe, 1975) show absorption across the rumen and a
net secretion of chloride in the omasum (Edrise et al., 1986). This
increase in concentration in the omasum does not appear to be related
to backflux from the abomasum but rather a permeability of the omasal
epithelium (Engelhardt and Hauffe, 1975).

The greatest absorption of sodium and chloride is through the
intestine (see Transcellular Sodium and Chloride Absorption by the
Small Intestine). Sodium balance is maintained by aldosterone and
deoxycorticosterone action on the convoluted tubules of the kidney.
Aldosterone promotes retention of sodium ions and secretion of
potassium in the urine. Normal blood sodium concentration in cows and
calves range from 260-280 mg/dl and normal chloride values are 101-113

mEq/L (Georgievskii, 1981).

Transcellular Sodium and Chloride Absorption by the Small Intestine

Sodium absorption may be accomplished by three processes. These

are: 1) active absorption not coupled to absorption of other solutes
but associated with passive chloride absorption (electrogenic), 2)
absorption coupled with transport of nonelectrolytes and 3) neutral

sodium chloride absorption (Schultz, 1981).

19

Electrogenic sodium transport is postulated to occur as fbllows
(Figure 1). Sodium absorption across the apical membrane occurs due to
the differences in chemical concentration (mucosal solution - 140 mM, 0
mV and cellular solution 15 mM and -40 mV) and electrical potential.
Sodium extrusion across the basolateral membrane in to plasna or
serosal solutions is energetically unfavorable (serosal sodium 140 mM
and + 3 mV) and is therefbre coupled to ATP hydrolysis. This is the
role of the Na+/K+-ATPase enzyme complex faund in the basolateral
membrane of most all animal cells. The diffusion of chloride is
coupled to the electrical potential difference generated by the active
absorption of sodium and the positive difference between mucosal and
serosal solutions (+3 mV). This diffusion occurs across the ”leaky"
epithelium.

Coupled sodium absorption (i.e., organic solute absorption coupled
to sodium uptake) is the proposed mechanism to account far D-hexose,
L:amino acid, di and tripeptide, water.soluble_vitaminand bile salt
absorption (Figure 2). The downhill movement of sodium across the
apical membrane energizes the uphill movement of solutes across
"carrier mechanisms" located in the mucosal membrane. The Na+/K+-
ATPase mechanism actively extrudes sodium across the basolateral
membrane maintaining low intracellular sodium activity which allows
continued entry of organic solute and maintaining the electrochemical
potential for sodium across the mucosal membrane. Solute transport
from the cell solution to the serosal solution occurs down a
concentration gradient. Evidence presented by Bihler and Cybulsky

(1973) and Danisi et a1. (1976) using intact intestinal preparations

20

Figure 1. A Model for Electrogenic Sodium Transport

 

 

 

 

 

No No
(140 mu 1“ (140nm
Ouooom
Omv - 40ml! +3 «11/.
c: «cg—I .- ::::::‘-’:: : 113—- c:

 

(120 mM) S; 1 1120mm
1

Schultz , 1981

21

Figure 2. Cellular Model for Na-coupled Absorption of
Organic Solutes (S) by Small Intestine

 

 

 

 

 

OrnV - 40 mv
No
S C) ——————————————— -
D- hexoses :—
L- amino acids .
dipeoh‘des
tnpeoiides

water sol vitamins
bile salts (ileum)

Schultz . 1981

22
and Murer et al. (1974) using isolated membrane vesicles indicate the
presence of membrane carriers on the basolateral membrane to facilitate
sugar and amino acid diffusion into serosal solutions. This process is
independent of sodium transport.

In summary, organic solute transport into the epithelial cell is
mediated by sodium transport in two ways. First the concentration
gradient that exists for sodium and second the electrical potential
difference created across the membrane by sodium transport.

Alterations of sodium transport might alter transport of organic
solutes or result in compensation by other mechanisms specifically the
Na+/K+-ATPase pump.

Several models have been pr0posed to account fbr sodium chloride
absorption. The first of these is shown in Figure 3a. Sodium chloride
is absorbed at the apical membrane by a one for one neutral mechanism.
The “downhill" movement of the sodium ion energizes the “uphill“
transport of chloride. Sodium is extruded through the basolateral
membrane by the Na+/K+-ATPase pump and chloride is tranported with
the energetically favorable gradient into the serosal solution. The
mechanisn of chloride extrusion is unknown (Frizzell et al., 1979).

A second model (Figure 3b) involves two antiport processes; a Na-H
exchange and a Cl-HCO3 exchange. The movement of sodium down the
concentration gradient at the apical membrane energizes the movement of
hydrogen from the cell against a gradient. This creates a basic cell
solution causing H003 movement out of the cell which generates energy
to pull chloride into the cell. Presumably sodium and chloride

transport into the serosal solution would occur much the same as is

23

Figure 3. Models for Chloride Absorption

 

 

 

 

 

 

 

MUCOSAL CELL SEROSAL
SOLUTION SOLUTION
Tia Cl 113

-—wv-cAMP
, a.
Cl HCO3 H30;C03&-—, CO:
r1
b.

a. Direct symport model

b. Dual Na-H and Cl-HCO antiport model

3

Schultz. 1981

24
postulated for the first model. This model would tightly couple sodium
chloride transport to intracellular pH regulation.
Copper

Absorption of capper has been shown to occur in the large
intestine (Stevenson and Unsworth, 1978) in sheep fed either fresh
forage or hay concentrate diets. In this study there was net secretion
of copper in the abomasum and forestomach, and no net absorption
between the duodenum and ileum. These data are contrary to those found
in calves or mature cows. In these studies (Chapman and Bell, 1963;
Ivan and Grieve, 1976), the duodenum, jejunum and ileum contributed
roughly the same amount of absorption. Secretion of copper in these
studies was shown to occur primarily befbre the duodenum (most likely
the abomasum) and along the small intestine. In the calves and mature
cows the large intestine did not absorb appreciable quantities of
copper.

The mechanisn of copper absorption is largely uninvestigated.
There is some evidence that c0pper may be absorbed in a passive process
(Bremner and Davies, 1980) or in a saturable transport system
(Neethling et al., 1968, as cited by Bremner and Davies, 1980). The
disparity in evidence may be due to the concentration used. At low
doses a saturable process seems to be indicated while at higher doses a
passive process appears to fit the evidence.

The factors that appear to affect capper absorption are: 1) the
chemical form of copper in the digesta and at sites of absorption, 2)

the level of molybdenum and sulfur in the diet, 3) cadmium and zinc

25
levels in the diet, 4) the protein level of the diet and b) the
presence of a protozoal population in the rumen. Bremner (1970)
examined the changes in the forms of zinc, manganese, and copper in the
intestinal tract of sheep. His data indicate that soluble copper
concentrations increased when digesta moved from the rumen to the
ileum. The solubility of copper was less than 20% in the rumen and
this was attributed to binding the insoluble rumen residue. Copper
solubility in the abomasum was very low. As the pH increased over 6 in
the intestinal tract, capper became increasingly soluble. In this
study Bremner was unable to determine what coppgr_was complexed with
and how the presence of a copper complex would affect absorption.

Molybdenum and sulfur are known to affect copper availability.
Molybdenum-copper complexes appear to be absorbed through the intestine
and transported through the blood and are excreted through the urine
and bile (Marcilese et al., 1969) as a complex. Copper-sulfur
interactions have also been shown to decrease copper absorption through
the fOrmation of insoluble copper-sulfidemcomplexes (Huisingh et al.,
1973). When molybdenum, sulfur and copper interact, copper
availability is further reduced. It is thought that thiomolybdate
derivatives may be formed in the rumen which interfere with copper
absorption (Bremner and Davies, 1980).

Cadmium and zinc levels in the diet are also thought to affect
copper availability. Indirect evidence implicates cadmium. In a study
with ewes and lambs, Mills and Dalgarno (1972) observed a decreased
copper retention with high levels of cadmium in forage. In studies

using bull calves, Ivan and Grieve (1976) have found decreased net

26
copper absorption with increasing zinc levels in the diet. This
inhibition was shown to occur in the shall intestine and rumen.

Ivan and Veira (1981) examined the effect of dietary protein level
on the solubility of copper in the rumen and abomasum of sheep. Their
data indicate that increasing dietary protein level from 7% to 19%
decreased copper solubility in the rumen from 23.5% to 15% (this was
not significantly different at P>.05). Abomasal copper solubility
decreased significantly (P<.05). The authors speculated in a later
paper (Ivan et a1, 1986) that this was due to degradation of soluble
protein to amino acids thereby increasing the availability of sulfUr
containing amino acids to bacterial and protozoal metabolism. This
metabolisn results in the production of sulfide which interacts with
copper to farm an insoluble complex.

In the same study the role of protozoa in copper availability was
examined. Rams were maintained either faunated or ciliate-free and fed
a high copper diet. The presence of ciliate protozoa decreased liver
copper accumulation 38-50%. This indicated a decrease in absorption of
copper. The mechanism by which the ciliate protozoa appeared to
alleviate a copper toxicity is related again to the degradation of
protein in the rumen and the availability of sulfur containing amino
acids to protozoal metabolisn.

Copper in the plasma is faund bound to cerulOplashin
(Georgievskii, 1981), and levels range from 8-120 ug/dl in the blood of
adult cattle, calves and sheep.

Zinc
There is disagreement as to where in the digestive tract of

ruminants zinc is absorbed. Miller and Cragle (1965) reported that 35%

27
of dietary zinc was absorbed between the mouth and abomasum.
Secretions of zinc in the intestinal tract (proximal duodenum) in this
study amounted to 280% of intake. There was also a snall amount of net
absorption throughout the shall intestine. Work by Grace (1975)
indicates that zinc is secreted rather than absorbed in the forestomach
of'rdminants, and that the small intestine, particularly the upper
segment is the primary site of zinc absorption. Other work utilizing
labeled zinc indicated that the rumen actively secretes zinc with
little absorption taking place. The abomasum was the primary site of
zinc absorption with about 40% of the zinc from rumen digesta absorbed.
Active absorption of zinc took place along the small intestine as did
secretion (Georgievskii, 1981). It seems that zinc is absorbed to some
degree from all segments of the tract from the rumen through the colon.
Zinc is also secreted from all of these segments. The small intestine,
however, is likely to contribute the greatest amount of absorption
(Bremner and Davies, 1980). -

The mechanism of zinc absorption across the mucosal cell is not
known. There is some evidence that zinc, like other minerals,
interacts with a protein molecule at the apical membrane which after
chelation enhances transport (Freeman et a1, 1971 as cited by Ashmend
et al, 1985). In any case, zinc absorption into the mucosal cells is
rapid, but passage into the blood stream is slow (Georgievskii, l98l).
Zinc in plasna is found loosely bound to albumin (transport) and
tightly bound to globulins. Normal plasma zinc levels in whole blood
range from 250-500 ug/dl in dairy cattle to 200-300 ug/dl in calves

(Georgievskii, 1981). Zinc excretion is through intestinal secretions,

28
pancreatic juices and saliva. There is little zinc in urine
(Georgievskii, 1981).
Selenium

Selenium is absorbed in the lower segments of the small intestine
of sheep and is secreted in small amounts through the intestine
(Georgievskii, 1981). Absorption is a rapid process that moves against
a concentration gradient. Data indicate that selenium is absorbed as
Se-amino acid complexes by the same mechanism as sulfur-containing
amino acids (Georgievskii, 1981). Selenium in blood is carried by
albumin and globulins and enters the red blood cell rapidily via an
oxygen-dependent active tranSport system. Normal blood selenium levels
in cattle are .09-.1 ug/ml.

Selenium excretion occurs through the kidneys, gastrointestinal
tract and lungs. The primary routes in ruminants are feces and urine
(30-35% each) and exhaled air (2-3%). As selenium intake increases the
urinary portion of selenium excretion increases (Georgievskii, I981).

Lipid Absorption

Lipid absorption in the ruminant animal begins in the rumen with
short-chain fatty acids (SCFA) transported across the ruminal
epithelium. The ability of the ruminal epithelium to transport SCFA's
is inversely proportional to the chain length. The order is butyric >
propionic > acetic (Sutton et a1, 1963). To some extent this process
is pH dependent with a decrease in pH increasing the overall rate of
absorption (Sutton et a1, 1963) suggesting that the epithelium is more
permeable to undissociated acid fonns (Stevens, 1970).

These ruminally absorbed SCFA's, or volatile fatty acids (VFAs),

are transported into the portal vein with a very minor amount absorbed

29
into the lacteals and transported through the lymphatic system (Kiddle
et al., 1951, Annison, 1965). SCFA's may also appear in the portal
blood from omasal (Gray et a1, 1954) and abomasal (Johnston et al.,
1961) absorption.

Long and medium chain fatty acids are also absorbed prior to the
small intestine (Wood et al., 1963) but the quantitative significance
of this process appears to be limited (Noble, 1981). The composition
of digesta found in the various points of the gastrointestinal tract
have been found to vary markedly from that found in the rumen (Lennox
et al., 1968). Digesta present in the rumen, abomasum and upper
duodenum have been found to be similar in composition (Bath and Hill,
1967; Lennox et a1, 1968) and distribution of lipid, despite changes in
diet composition. In this area of the tract, digesta is comprised of
unesterified fatty acids, small amounts of phospholipids and very
little triglyceride. The unesterified fatty acids were fbund to be
closely associated with the particulate fraction while phospholipids
were more evenly distributed with the aqueous and particulate phases
(Smith and Lough, 1973).

In the lower duodenum and jejunum the majority of the lipid is
largely esterified. Rumen and abomasal lipid consisted of stearic acid
primarily while jejunal lipid contains a greater proportion of
phospholipids and esterified fatty acids. This source of lipid has
been found to be bile lipid secretions.

Johnson et a1. (1974), in a study investigating the site of lipid

absorption in the sheep intestine, showed limited hydrolysis of

30
esterified fatty acids and 20% of the fatty acids were absorbed
unesterified from the upper jejunum. A further 60% of the total lipids
were absorbed from the middle and lower jejenum. These absorbed acids
were derived from the hydrolysis of phospholipids and neutral lipids.
By the ileum, lipid absorption was almost complete.

The mechanisn of lipid absorption in the ruminant is much the same
as that for the monogastric, although the ruminant has been shown to be
very efficient at lipid digestion and in fact has a greater ability to
digest dietary fat than does the monogastric (Carroll, 1958; Carroll et
al., 1958; Andrews and Lewis, 1970).

The major steps in mucosal uptake of dietary lipid include: 1)
intraluminal digestion, 2) difosion across the unstirred water layer,
3) membrane permeation, 4) metabolism and lipOprotein assembly, 5) exit
from the mucosal cell (Thomson and Dietschy, 1981). Digestion proceeds
in the small intestinal lumen with the interaction of‘bile and
pancreatic secretions. In the ruminant the pancreatic duct and bile
duct empty into the duodenum through a common channel. This process is
a continuous one because of the relatively constant flow of digesta
entering the duodenum (Noble, 1981). The absorption rate of digested
lipid is controlled by the rate of diffbsion across the unstirred water
layer and penetration through the apical membrane (Thomson and
Dietschy, 1981). The unstirred water layer (UNL) consists of a series
of water ”layers" each becoming progressively more stirred until it
blends with the water phase. Transport of molecules across this layer
is dependent on the thickness of the URL, the diffusion constant of the

molecule to be transported and the concentration gradient present

31

between the bulk phase (luminal lipid) and the cell membrane (Thomson
and Dietschy, 1981). It is at this point that bile acids.and bile salt
concentrations play a critical role in the absorption of lipid across
the apical membrane of the mucosal cell. Micelle formation is
dependent on bile salt concentration. Unless a critical level of bile
salt/lipid is reached micelles will not form. Since long chain fatty
acids and monoglycerides have low water solubilities, and since the
fbrmation of micelles may increase solubility of lipid 100-1000 times
(Higgens and Barrnett, 1971), diffusion across the URL is increased by
micelle fbrmation. However, there is also, an increase in diffusional
resistance with increased size of the bile acid-lipolytic monomer but
this is overcome by the great increase in the concentration gradient
(Higgins and Barrett, 1971). This is perhaps better seen using the
following equation (Thompson and Dietschy, 1981) that describes
movement across the URL.

J = C(Cl-C2)D7d where,

J = rate of movement of solute

C1 = concentration of solute at the bulk phase (digesta)

C2 = concentration of solute at the aqueous - membrane

interface

0 = diffusion constant of the molecule

d = functional thickness of the URL.
If (C1-C2)D is increased by solubilization and d is constant, then
J will increase.

There are three theories that may help explain the events that

occur during lipid uptake from the micelle into the membrane (Thomson

32
and Dietschy, 1981). One is that the micelle simply is taken up in a
pinocytosis-like action. This appears to be unlikely because
experiments in which uptake rates of various fatty acids were measured
indicated that the constituent parts of the micelle were absorbed at
rates independent of each other (Hoffman, 1970; Hoffman and Simmonds,
1971). The second possibility is that the micelle interacts with the
membrane in a collision which excludes water. The fatty acids within
the micelle moves into the cell and the bile acids return to the URL
(Thomson and Dietschy, 1981). Experimental evidence does not fit this
model (Hestergaard and Dietschy, 1976). In the third model, which best
fits the evidence, the micelle acts as the solubilizer from which
molecules pass into the microvillus membrane (Nestergaard and Dietschy,
1976). Actual uptake of the fatty acid is determined by the
concentration in the micelle which is in equilibrium with the fatty
acid concentration in the aqueous solution (Hestergaard and Dietschy,
1976). '

Once inside the mucosal cell the resynthesis of triglyceride
occurs primarily through the a-glycerOphosphate pathway (Noble, 1981).
Phospholipid resynthesis mechanisms are not well defined in the
ruminant intestine, but the evidence suggests that the major pathway is
the reacetylation of 1-lysolecithin (Subbiah et al., 1969).

Cholesterol is produced primarily by the small intestine in the
ruminant (Scott and Cook, 1975) in a process which is sensitive to
changes in diet and other factors (Scott and Cook, 1975).

Following resynthesis within the smooth endoplasmic reticulum, the

absorbed or synthesized lipid is incorporated into chylomicrons or

lip0proteins (VLDL's) and is secreted from the Golgi apparatus in

33
secretory vesicles. These vesicles migrate toward the basolateral
membrane where reverse pinocytosis is thought to occur. Microtubules
are thought to have an integral role in this process (Barrowman, 1984).
The chylomicra then pass through the lamina pr0pria to the lacteal.
This process is not clear. There is evidence that the chylomicra pass
through interendothelial gaps (Casley-Smith, 1962) into the lymphatic
endothelium. Once inside the lacteal the lipid moves through the
intestinal lymphatic system until it is eventually released into the
plasma through the lymphatic duct (Noble, 1981).

The flow of lymph through the lymphatic system has been shown to
be related to the amount of fluid absorption through the intestine,
elevation of portal venous pressure and increased plasma dilution
(Barrowman, 1984). In general an increase in intestinal fluid in the
mucosa increases fluid pressure which stimulates lymph flow. In the
ruminant animal there is virtually continuous lipid absorption and
lymph flow under normal dietary conditions (Noble, 1981).

Harrison et al. (1974) examined the composition of lymph from
sheep fitted with thoracic duct catheters. Seventy-three percent of
the lipid present was associated with the VLDL fraction and 27% with
the chylomicrons. The major classes of lipid present were
triglycerides, phosopholipids and cholesterol with triglycerides making
up the greatest amount. The relative proportions of the lipid classes
do not appear to be altered with different dietary regimes, but the
concentrations of triglycerides, phospholipids and cholesterol in
thoracic lymph as well as lymph flow rate may be altered (Noble,

1981).

34

The distribution between lymphatic lipid absorption and direct
portal absorption is known to be in favor of lymphatic absorption.
Generally speaking, long chain fatty acids are incorporated into
triglycerides and are transported to the lymphatic ducts as
chylomicrons (McDonald et al., 1980). There is evidence in sheep that
in the absence of bile, a substantial amount of the lipid absorbed does
not appear in the lymph (Noble, 1981). This suggests that lipid
absorption through the portal vein might be of more importance than
lymphatic absorption in the ruminant. However, in later absorption
studies with labeled fatty acids, no labeled acid could be detected in
the jugular blood casting doubt on this hypothesis (Harrison and Leat,
1972).

Glucose Absorption

The ion gradient hypothesis of coupled sugar-sodium absorption was
first proposed by Crane (1961). His hypothesis was that the energy
required fbr establishment of a concentration gradient for sugar
transport is derived from the flow of sodium ions down a concentration
gradient into the cell. He also postulated that a membrane carrier or
transport site was involved with sugar absorption and that this carrier
had different affinities fbr sugar depending on the presence or absence
of sodium. Since 1961, evidence has mounted that confirms this
hypothesis (Kimmich, 1981).

The sodium that is absorbed is extruded across the basolateral
membrane via a ouabain-sensitive energy dependent mechanism (Schultz,
1981). This mechanism functions to maintain low concentrations of

sodium intracellularly and also acts to maintain the electrochemical

35
potential difference for sodium across the apical membrane (Schultz,
1981).

Use of membrane vesicles has confirmed Crane's sodium-dependent
glucose transport hypothesis and has also provided evidence that there
is another pathway of glucose transport across the cell (Stevens et
al., 1984). In membrane vesicles from steers and rabbits (Kaunitz et
al., 1983) the so called diffusive pathway was examined. This pathway
exhibits first-order kinetics between 0 and 20 mM glucose. It is also
evident from these studies that there are two saturable transport
systems. One of these is a high affinity system that predominates when
glucose concentrations are (.5 mM. The other is a high capacity system
that predominates at higher concentrations. Above 2 mM the diffusive
pathway is most important (Stevens et al., 1984).

Data from steer membrane vesicles indicate that sodium acts as a
competitive activator in the steer. That is, sodium affects Kt (half
maximal transport) but not Jmax (maximum rate of transport) (Stevens et
al., 1984). Other work from bovine membrane vesicles indicates that
the coupling stoichiometry of Na+:sugar in the sodiunglucose
high-capacity system was 1:1, while the low capacity, high affinity
system had a ratio of 3:1 (Kaunitz and wright, 1984). This is in
agreement with workers using kidney preparations (Stevens et al.,
1984).

Glucose exit through the basolateral membrane has also been shown
to function via a saturable facilitated diffusion system (Wright et
al., 1980). This system has been shown to be inhibited by structural

analogs (galactose) and inhibitors (cytochalasin B and phloretin) and

36
exhibits stereospecificity and saturation kinetics (Wright et al.,
1980).

The capacity of the bovine intestinal tract to absorb glucose
actively has been questioned. Work done by McAllen and Lewis (1985)
and Pehrson et a1. (1981) indicates that when glucose is presented to
the small intestine it is absorbed with an efficiency > 80%, However
when starch is presented to the small intestine the efficiency of
absorption is reduced (51%). These authors found that while the starch
was well decomposed, its end products for absorption were not utilized
as well as glucose. Their conclusions were that at high levels of
starch, maltase activity of the small intestine is inadequate to
process all of the starch (Pehrson et al., 1981) and that starch
digestion and utilization is different depending on the type of starch
used (McAllan and Lewis, 1985).

Amino Acid and Peptide Absorption

Dietary proteins are hydrolyzed to amino acids and small peptides.
Both of these farms of'degraded protein are transported across the
intestinal brush border although through different mechanisms.
Ganapathy and Leibach (1982) provided a summary of the various features
known about peptide transport. These include:

1) Most amino acids are absorbed faster as peptides than as free

amino acids

2) There appears to be no direct competition at the mucosa between

amino acids and peptides fbr transport

3) Sites of maximal absorption of amino acids and peptides in the

small intestine are likely to be different

37
4) Peptide absorption may occur either by hydrolysis at the brush
border by peptidases present there and entrance to the cell as
a free amino acid or by special systems in the membrane that
absorb peptides intact.

Other details of peptide absorption remain to be elucidated. The
dependence of peptide transport on Na+ concentrations or the sodium
gradient in the membrane has been investigated but the results vary
considerably (Ganapathy and Leibach, 1982; Adibi and Kim, 1981 and
Alpers, 1985). In some studies Na+ is essential while in others it
is not. Studies investigating the area of the intestine with the
greatest ability to transport peptides have resulted in the conclusion
that the jejunum and ileum do not show differences in peptide transport
disappearance rates (Adibi and Kim, 1981).

Recently Ganapathy and Leibach (1985) proposed a proton gradient
energized peptide transport system. These investigators hypothesized
that a NaT/H+ exchanger mechanism propels H+ ions into the lumen
where a coupled peptide - H+ transporter moves the peptide-H+
complex into the cytosol through a proton gradient. Nhile this
hypothesis may have merit and does explain some of the evidence
presented in peptide absorption studies, it remains to be validated.

Still unclear is the extent to which peptides are hydrolyzed by
brush border amino peptidases and absorbed as free amino acids or
absorbed as peptides.

Amino acid transport has been better elucidated. Niseman, over 25
years ago, established that amino acids were transported uphill (Munck,

1981). Later evidence established that transport of some amino acids

38
was related to and energized by the maintenance of low intracellular
sodium concentrations and high intracellular potassium concentrations.
Eventually Crane (1977) formulated the sodium gradient hypothesis that
fit both glucose and amino acid transport data. Inhibition studies
using cardiac glycosides further established the dependence of amino
acid uptake on the Na+/K+-ATPase located on the basolateral
membrane of the cell membrane (Csaky, 1963).

Not all of the amino acids transported across the cell are
dependent on the sodium gradient (Stevens et al., 1984). The N83 (most
neutral amino acids), IMINO (glycine and imino acids), PHE
(phenylalanine, methionine), A (short chained polar amino acids) and
ASC (3-4 carbon neutral amino acids, alanine, serine and cysteine) are
sodium dependent. The y+ (lysine, arginine, cationic amino acids) and
L system (leucine, branched and ringed amino acids) are not sodium
dependent (Christenson, 1982; Stevens et al., 1984). .

The most active site of amino acid absorption in sheep was found
to be mid to lower ileum. The area of the greatest rate of absorption
of lysine was faund to be the jejunum (Johns and Bergen, 1973).

Monensin

Much work has gone into elucidating the mechanism of action of
monensin in the rumen. Several reviews have been written on the
subject (Bergen and Bates, 1984; Schelling, 1984). In summary,
monensin shifts the acetate-propionate ratio toward increased
propionate. Gram positive bacteria are inhibited and methane and
fumarate production are reduced. The incidence of lactic acidosis and

ruminal ammonia concentrations are reduced. Animal perfbrmance is

39
enhanced as indicated by decreased feed intake with no reduction in
rate of gain (Goodrich et al., 1984).
Metabolism of Monensin

Several studies have been conducted to examine the metabolism of
monensin in the steer, chicken, horse, pig and rat (Herberg et al.,
1978; Donoho et al. 1978; Davison, 1984; Donoho, 1984). In a study
with (”Cl monensin, 94% of the radioactivity from the dose of
monensin was recovered in the feces (Herberg et al., 1978). Further
investigations by Donoho et a1. (1978) resulted in the identification
of 4 metabolites in steer feces. Ninety percent of the 57% of the
recovered radioactivity was identified by mass spectroscopy to be
monensin. The remaining 10% was identified to be 4 O-demethylated
fragments of monensin. Labelled monensin was also incubated in rumen
fluid (12 hr), abomasal fluid (5 hr) and cattle feces (2 days) to
examine alimentary degradation. In all cases at least 90% of the
labelled compound was recovered.

Biliary metabolism of monensin has also been examined (Davison,
1984; Donoho, 1984). Thirty to fbrty percent of a labelled monensin
dose was absorbed in calves. Thirty-feur to thirty-seven percent of
the radioactivity was recovered in the bile, 50 to 64% in the feces and
trace amounts in gallbladder, GI tract, liver, kidneys and carcass
(Davison, 1984). These data agree with Donoho et al. (1978) who
reported absorption of approximately 50-60% of labelled monensin in
steers. Of this label only 3% was fbund to be monensin. The rest was
in the form of O-demethylated derivatives of monensin. The biological

activity of these derivatives was also examined. In several systems

40

evaluating antimicrobial activity, anticoccidal activity, ionophoric
activity and cytotoxic activity the metabolites were found to be 20
times less active than the parent compound (Donoho, 1984).

These data would indicate that ruminant animals absorb
approximately one half the dose of monensin and that animal metabolism,
not gut bacterial action, is responsible for monensin metabolism.

Monensin's Effect on the Mai/K+-ATPase

Monensin, a carboxylic ionophore (see Figure 4), has been used in
many studies and with many cells and tissues as a cellular perturbant.
By virtue of its ability to collapse proton and sodium gradients across
cell membranes, monensin is a valuable tool in examining the role of
membrane gradients on various cellular functions. Table l is a
compilation of many of these studies. The cellular effects of'monensin
range from reduced secretion of products from the Golgi complex to
increased secretion of catecholamines from chromaffin cells. Other
effects include disruption of post-translational modification of
membrane and secretory proteins, inhibition of various phases of
endocytosis, intracellular pH alterations and undersulfation of
chondrocytes and chondrosarcomas (Ledger and Tanzer, 1984).

The majority of these studies summarized in Table 1 use the
ionophore, monensin, as a means to investigate sodium-dependent
membrane processes. The mechanism by which monensin disrupts ion
transport is described by Pressman (1976) and Bergen and Bates (1984)
and is illustrated in Figure 5. Monensin in a protonated form diffuses
to one membrane interface and releases its proton. This increases the

polarity of the compound and traps it at the polar interface of the

41

Figure 4. Schematic Views of the Structure of Monensin
and Monensin Sodium (Pressman. I976)

 

CH3
E C“;
CU’O O ‘ O CH1OH
‘ ' HO
00°C (”3 Monensin
CH3
3 A
.0 C 0
0 0
0 ' j
C . . 0' H
.'0 o'. H t
i
i I
0'. . (E: c.)
D :
O
E ‘H
O
OCH} .

Monensin sodium

2
4

 

mwop .._e be seed
amma_ .._a so co_;.m
ammo. .._e ea ca_;_m

ewmp .._m um memncmceoz
«mop .._e um amasscm

ewe. ..P. be amen

ewe, .._e “a oeexee

Nmm_ .cmNcm» use eemz_wmx
Name .._a “a aoeeegm_x

swap ..F. be opeesoo

News .ceeceo eee oe..u

ea_o

toumcmmu :_muoeq umuwnwscfi
peoamcecp Lemzm mmuepasvpm
utoamcocu Leona mmuepsewum

wxmuaz swoon—m commmcucfi
In uvpomouxu ummmmcomo

meccmumwu =m__ozm

mmea~cm _msomom»_coc new

meOmomx_ Co mmmmpwm

mcmux_mo=.5emouxpm enema—xxo

aim yo cowue_=e=uu<
cowuumcucou mmxe—ma

mwmmgpcaM cwmuocq mp_aw;c~

mcwmmmuota
oeoa_aoaeca accesses

mmeu_e:v5emouzpm
toiuupxumouiz
amouapm_»:umEto-m
wmouzpmpazumEIOim
amouz—m_»;umeio-m
amouapmaxomuiu

mmeuwmou=_miu
mmmcwcoesua—miu
mcwEmmonPmfiuara
.mcrtmm arm;

ae<
=_peouocepmeorqoiocq

cwucooocmmeo_QOioca

mweomomxg Lm>w4 pom
Emmcgaewv mmzoz
mmuxuoccuagm cmw><

mmuxooexgp mmaoz

m__mu mPeLmuonge uooe mNWez

mzueemgam _m—ou acepa
momezaocuee
_mmcouwcma mmaoz

mmuxuocvcogu xu_;u
__ou «Peony meg em:_=w

scea_=aaa See

xeea.=u_a See

 

owa_ .oeem=< eee £5.2m asea +¥\ez obesaaoq m~< men
mem_ .acemeoooa ace eSPEm ease +x\+<z obesaeo< +amem+az- men
mucmcmwmm cowpo< museumnzm acme .pmu

 

 

“cancsucma

Lepsp—wu a we cpmcmcoz mcw>—o>:fi mmwvaum we xgm553m <

.p mpnoh

43

cell membrane. Nhen a complexable cation interacts with the anionic
ionophore, a complex is formed and the zwitterion complex is able to
move away from the membrane and diffuses to the opposite interface.
Here the cation is released and the anionic ionOphore is free to become
protonated and then to continue the proton-cation exchange cycle.
Carboxylic ionophores act as exchange diffusion carriers because their
final equilibrium is independent of the membrane potential but is
sensitive to pH (Pressman, 1976). Monensin has the greatest affinity
for sodium ions with decreasing affinity fbr potassium, rubidium,
lithium and cesium (Pressman, 1976).

Smith and Rozengurt (1978) used monensin as a sodium perturbant in
a study investigating the activation of quiescent fibroblasts (3T3
cells). These data indicate that the Na+/K+-ATPase present in
those cells is limited by internal sodium concentrations and extremely
sensitive to small changes in internal sodium. Monensin (at 4 ug/ml
media) increased internal sodium concentrations three fald in 30
minutes. Monensin at all concentrations stimulated Na+/K+-ATPase
activity, although 3 ug/ml media produced maximal stimulation. Ouabain
(at lmM) inhibited monensin's stimulation of the pump. Low
concentrations of'monensin were shown to stimulate a slight increase in
cell potassium. Higher levels of'monensin caused a net efflux of
K*.

Further work by this group (Smith and Austic, 1980) demonstrated
an increased a-aminoisobutryic acid (AIB) uptake with a monensin
induced stimulation of the NaT/KT-ATPase pump in the 3T3 cells.

A18 is the non-metabolizable substrate for the Na-dependent A system

44

Figure 5. A Schematic View of the Mechanism of
Monensin's Action in the Cell Membrane

 

MEMBRANE

 

Carboxylic ionophore mediated cation
transfer across a bimolecuiar lipid membrane. M’
. metal cation: I - ionophore: H’ . proron, I-I-I I
proconated ionophore: M’I' s zwitterion of metal
cation and anionic form of ionophore

Pressman, 1976

45
absorption (Kilberg, 1986). Monensin, at 15 pH, increased the initial
rate of A18 uptake as well as the steady state level of A18 at least
3-f01d. Ouabain (lmM) which had no effect on AIB uptake without
monensin, inhibited monensin stimulation of AIB uptake. Again monensin
increased sodium concentration in the cell but there was no increased
potassium.

Nordenberg et al. (1984) and Bihler et al. (1985a) have
demonstrated that monensin affects glucose uptake in mouse thymocytes
and mouse skeletal muscle. In the work of Nordenberg et al. (1985)
monensin (7.2 uM) significantly (P<.001) stimulated 2-deoxyglucose
uptake and 3-0-methylglucose transport in mouse thymocytes. Ouabain
did not inhibit monensin's stimulation of glucose uptake, but low
concentrations of sodium and cytochalasin B (an inhibitor of glucose
uptake that binds to the membrane receptor) did. Bihler et al. (1985)
faund an increase in internal sodium, calcium and 3-O-methy1-O-glucose
concentrations and a stimulation of the Na+/K*-ATPase with monensin
addition (12 ul monensin) to mouse diaphragm. Mitochondria isolated
from rat hind limbs indicated the monensin increased mitochondrial
calcium when calcium was present in the media. This is likely to be an
effect on mitochondrial Na+/Ca+-ATPase.

In avian erythrocytes (Bihler et al., 1985b), monensin-stimulated
sodium and 3-0-methyl-D-glucose uptake was dependent on increased
sodium influx. Monensin was also shown to decrease cellular ATP
concentrations reflecting increased Na+ pumping in the plasma

membrane.

46
Blood Flow Technique

Growth, lactation and pregnancy are demanding physiological states
for the ruminant animal. Much work has been done to examine the
metabolic processes occurring in the rumen and lower digestive tract
but less infbrmation is available about the amount of absorbed
metabolites reaching the liver. The splanchnic bed, the gut, and the
liver play key roles in regulation of nutrient availability. Several
techniques have been used to evaluate the amount of absorption through
the gut and hepatic metabolism. Two techniques have been applied to
the whole animal. These are based on venoarterial (VA) concentration
differences coupled with measurements of blood flow or isotope dilution
methods designed to measure entry rates in the whole body (Annison,
1965).

Techniques that evaluate V-A differences and turnover rates depend
on animals with many catheters. Catheters may be placed in the portal
vein, hepatic vein and an artery for sampling and mesenteric or
peripheral veins for infusions.

The techniques that utilize V-A differences to assess absorption
or metabolism rely on the direct measurement of blood flow rates or
assumption of blood flow. Blood flow may be measured by several means.
These include: thermodilution (Bensadoun et al., 1962, Webster et al.,
1975); radioactive microspheres (Shephard et al., 1984, Dinda et al.,
1983); Laser-Dappler, fluorescein or electromagnetic flowmetry (Perbeck
et al., l985a,b; Shepherd et al., 1984); hydrogen gas clearance
(Lundgren, 1980); iodoantipyrene clearance (Dugas and Hechsler, 1982);

dye dilution (Katz and Bergman, 1969; Huntington, 1982). Dyes such as

47

Evan's blue, indocyanine green, sulfobromophthalein (ESP) and
p-aminohippuric acid (PAH) are used most commonly and their use is
dependent upon the system to be investigated.

The most important consideration in blood flow studies is the same
as in any marker study, complete mixing (Katz and Bergman, 1969).
Blood samples are withdrawn very slowly over several minutes to insure
mixing of metabolites and dye (Katz and Bergman, 1969). Another
consideration important in blood flow techniques is insuring that the
dye is at steady state. This is done via a bolus infusion of dye in
the mesenteric vein for 15 minutes in sheep (Katz and Bergman, 1969)
and 10 minutes in cattle (Huntington, 1982) followed by continuous
infusion of dye for another 20 minutes befbre sampling (Huntington,
1982).

p-Aminohippuric acid is the dye used most commonly in blood flow
studies in cattle and sheep. Data presented by Huntington (1982, 1983)
and Huntington et al. (1981, 1983) have examined portal blood flow and
net nutrient appearance in the portal vein under various experimental
experimental conditions. These studies indicate that dietary
differences may affect portal blood flow estimates (Huntington, 1983),
net D-lactate absorption estimates (Huntington et al., 1981),
ammonia-nitrogen absorption estimates (Huntington, 1982), plasma urea
nitrogen absorption estimates and some amino acid absorption rates

(Huntington et al., 1981).

In Vitro Measurements of Nat/KT-ATPase Activity in Biopsies of
Intestinal Mucosa Taken From Duodenally Cannulated Beef Steers and

Heifers

48

49
Introduction

After examination of the available literature on monensin made of
action in ruminant animals, data on monensins fate in the animal and
data in which monensin is used as a cellular perturbant, it becomes
apparent that there is a possibility, in fact a likelyhood, that
monensins action goes beyond those seen in the rumen. Data of'Davison
(1984, Donoho (1984) and Herberg et al. (1978) indicate that at least
50% of monensin fed is absorbed through the small intestine tract
before fecal excretion. This observation coupled with numerous in
vitro studies in which monensin stimulates the Na+/K+-ATPase pump
in eucaryotic cells poses an interesting question. What is the effect
of monensin feeding upon the mucosal Na+/K+=ATPase in the
gastrointestinal tracth

In order to answer this question two studies were designed and
carried out. The first of these involved in vitro incubation of
mucosal tissue obtained with a suction biopsy device to_examine the
NaT/KT-ATPase activity with graded quantities of monensin. The
second involved feeding monensin to cannulated steers and heifers with

biopsies removed to measure Na+/K+-ATPase activity in vitro.

Materials and Methods
Biopsy Technique
Biopsies were obtained, from steers and heifers, using a suction'

biopsy device (Quinton Instrument Co., Seattle, Washington). The

 

biopsy tube was passed through a duodenal cannula and inserted into the

descending duodenum. Either one or two biOpsies were removed per cut,

50
washed in warm, air-saturated buffer and transferred to another vial of
buffer until oxygen consumption could be measured. Best results were
obtained with the biopsy instrument after the duodenal contents had
been allowed to drain out of the cannula for about 10 minutes.
Infusing air through the instrument with a syringe several times also
helped biopsy removal. This seemed to clear the duodenum and permitted
the instrument to move against the duodenal wall which allowed the
suction to be effective. It might also be noted that there seemed to
be a relationship between the size of animal and effectiveness of the
instrument. Small animals, 230-400 kg vs 725 kg, enabled more rapid
sampling. This might be due to the size of the duodenum with the
lighter animals having a smaller duodenal circumference than larger
animals.

Up to six individual biopsies per animal were removed at each
sampling time. Animals were sampled on two consecutive days and then
allowed to recover for at least four days. There was never any
evidence that taking biopsies altered the animals behavior, eating
patterns or healthgy

Handling of Biopsies

As stated previously, as soon as the biopsy was removed from the
instrument capsule or wire, it was washed in buffer and then 1
transferred to another buffer solution. The buffer used was
Krebs-Henseleit buffer (KH) (Table 2) containing 10 mM D-glucose and 20
mM Hepes. This buffer was kept at 37°C and was air saturated (700 mm
Hg, 180 nmol OZ/ml; Umbreit et al 1964). Biopsies were kept in this

buffer until oxygen consumption measurments could be initiated.

51
Oxygen Consumption Measurements

Excised biopsies were transferred to oxygen (02) electrode
chambers containing 4 ml of Krebs-Henseleit buffer (air saturated,
37°C). 02 consumption was measured polarographically using a YSI
(model 53) Biological Oxygen monitor. Initial oxygen consumption was
measured for 15 minutes (McBride, 1984). At this time biopsies were
transferred to chambers containing KH buffer and 2x10'3 M ouabain.
Further oxygen consumption measurements were made for 40-45 minutes in
order to measure ouabain-insensitive respiration. Biopsies were then
dried at 90’C for 12 hours to determine dry matter content.

The principles behind the operation of the oxygen monitor is that
the oxygen sensor (probe) measures oxygen pressure. The membrane
placed on the sensor is oxygen (gas) permeable. Oxygen in solution
diffuses through the membrane and is consumed by the sensor;
02+2H20+4e’=40H'. The consumption of oxygen generates a
current which is proportional to the amount of oxygen to which the
sensor is exposed (Yellow Springs Instrument Co. Inc.. Yellow Springs,
Ohio). .

Calculations

The difference between oxygen consumption measured initially and
that measured after ouabain addition was called ouabain-sensitive
respiration. This is the respiration attributable to the
Na+/K*-ATPase activity in the cell. A11 respiration values are

expressed as nmoles 02 consumed/mg biopsy DM/min.

52

Table 2. Modified Krebs-Henseleit Buffer1

 

 

 

Chemicals : Parts By Volume
Sodium Chloride (0.154 M) 100
Potassium Chloride (0.154 M) 4
Calcium Chloride (0.11 M) 3
Potassium Phosphate (0.154 M) 1
Magnesium Sulfate (0.154 M) 1
Sodium Bicarbonate2 (.151 M) 21

D-Glucose 10 11M

Hepes 20 mM

 

1Dawson et a1, 1969; pH = 7.4.

ZGassed with 100% coz for 1 hour before mixing.

53
Preliminary Experiments

The techniques used to measure oxygen consumption and
ouabain-sensitive respiration from duodenal biopsies of sheep and
cattle were developed by McBride and Milligan (1984). In order to
revalidate some of their results and to insure that the procedures
would work in our hands, several preliminary studies were conducted.

Two Angus heifers (225 kg and 250 kg) were fitted with duodenal
cannulas. These cannulas were made of silastic tubing (Weber and
Rumpler, 1983) and inserted approximately 10 cm from the pylorus.

After a week of healing, the heifers were examined endoscopically and
then a biopsy was excised using the suction biopsy device available to
us courtesy of Dr. Thomas Herdt, M.S.U. Veterinary Clinic. The
biopsies excised were of adequate size to measure in the oxygen monitor
and were reasonably easy to obtain. At this time we attempted to
estimate the degree of damage to the duodenum at the biopsy site but
there was too much bleeding to see the site. Several days later the
site was unidentifiable and there was no evidence of damage to the
duodenum. There appeared to be no evidence of any discomfort to the
animal during the procedure and no subsequent alterations in feed
intake or behavior.

The heifers were placed on a high moisture corn:ground hay diet
(Table 3) and were accustomed to the metabolism room. Biopsies were
removed, and a dose response curve was constructed. Maximal inhibition
of respiration was achieved at lxlO'3M ouabain. This is similar to
McBride's data for cow duodenal biopsies, although in his data, maximal
inhibition was achieved at 1xlO’5M as well as 1x10'3M ouabain.

This data are in agreement with those of Liberman et al. (1979) for rat

54
jejunal mucosa. All further studies used concentrations of 2x10’3M
ouabain to inhibit respiration. It should be noted that actual
inhibition of NaT/KT-ATPase-dependent respiration was not directly
measured so these data represent lesser estimates than those that would
have been observed to all enzyme units were exposed to ouabain.

The length of time that the biopsies remained viable (i.e., steady
oxygen consumption traces) was also examined. It was found that the
biopsies remained viable for approximately 70 minutes in ouabain-free
media. This is in agreement with McBride and Milligan (1984) who found
duodenal biopsies from cows viable for 70-80 minutes in ouabain-free
media. Therefore, when a biopsy was removed, the time at which it was
placed in buffer was recorded and any biopsy older than 20 minutes was
not used. This occasionally occurred in transit from the Beef Cattle
Research Center to the laboratory (especially if there was a train), or

in situations when there was difficulty in obtaining another biopsy.

55
Study 1: In Vitro Addition of Monensin to Duodenal Mucosal Biopsies
This study was designed to answer the question, does addition of
monensin to duodenal biopsies taken from beef cattle affect
ouabain-sensitive respiration (Na+/K+-ATPase). The hypothesis was
that measured respiration rates with monensin additions of l, 5 and 10
ppm would not differ from biopsies with no added monensin.
Animals
Two sets of beef cattle were used in a study designed to

investigate the effect of monensin addition in vitro. One set was two
Angus heifers (225 kg and 250 kg) and the other was two Simmental
crossbred steers (300 kg and 318 kg). All animals were fitted with
duodenal cannulas inserted approximately 10 cm from the pylorus. These
cannulas were made of silastic tubing (Weber, 1983) and were
exteriorized just distal to the last rib in both heifers and l steer
and between the 12th and 13th rib in the other steer. .All animals had
been cannulated at least 2 months prior to the beginning of this study
and were well adjusted to their diet. Animals were housed at the Beef
Cattle Research Center in the metabolism room and had ad libitum access
to feed and water. Feeding was done twice daily at 8:00 AM and 6:00
PM. Lighting was continuous and daily exercise in a holding pen was
permitted.

Diets

 

The diet fed to the heifers is presented in Table 3. The diet fed
to the steers is shown in Table 4.
Sampling

Biopsies were obtained as previously described. Each animal was

sampled twice for each concentration of monensin examined.

56

media

Krebs-Henseleit buffer (Table 2) as described previously was used.
Monensin concentrations examined were 0, 1, 5 and 10 ppm which were
equivalent to O, 1.49 pH, 7.46 uM and 14.9 uM. All bioosies were
preincubated 10 minutes in buffer containing appropriate monensin
concentrations before oxygen consumption measurements were taken.
Monensin was provided courtesy of Dr W.G. Bergen.
Analysis

Data were analyzed using a one way analysis of variance.
Contrasts were made using a T test. The contrasts examined were:
Control versus monensin supplementation, 1 ppm monensin versus 5 and 10

ppm monensin and 5 ppm versus 10 ppm monensin.

57

Table 3. Composition of Heifer Rations

 

 

 

Feed . ,kg DM/day
High Moisture Ground Ear Corn 4.44
Ground Hay 1.60
Supplement1 ._;65
6.69

 

1Supplement contained soybean meal (94.6%), trace
mineral salt (2%), limestone (1%), selenium 90 and

Vitamins A, D and E.

58

Table 4. Composition of Steer Rations

 

 

Ingredient kg DM/day
High Moisture Ground Ear Corn 6.24
Ground Hay 2.11
Supplementl _;68
9.0

 

ISupplement contained soybean meal (94.6%), trace
mineral salt (2%), limestone (1%), selenium 90,

Vitamins A, D and E.

59
Study 2. The Effect of Monensin Supplementation 0n Duodenal Mucosal
Na+/K+-A1Pase Activity

The purpose of this study was to determine whether supplementation
of monensin (RumensinTM) to beef cattle diets altered
Na+/K+-ATPase activity in the duodenal mucosa of heifers and
steers. The hypothesis was that monensin addition (300 mg/hd/day) had
no effect on NaT/K+-ATPase activity as measured by in vitro oxygen
consumption. 1
Animals

The animals used were two Angus heifers (225 kg and 250 kg) and
two Simmental crossbred steers (300 kg and 318 kg). All animals had
been previously fitted with duodenal cannulas approximately 10 cm from
the pylorus. Cannulas were made from silastic tubing according to
Weber (1983). The cattle were housed in the metabolism room at the
Beef Cattle Research Center. Water was available ad libitum; and feed
was provided twice daily. Heifers and steers were allowed daily
exercise and lighting was continuous.
Leash

The design of this study was a switchback with each animal serving
as its own control. Treatments were no added monensin or monensin
(RumensinTM) fed at 300 mg/hd/day. The trial was run in two
replicates. The first was with the heifers and the second fallowed
approximately 6 months later with the steers. Each animal was sampled
twice (8 total biopsies) per treatment. The cattle received
RumensinTM at full dosage for at least 10 days prior to sampling
and were removed from RumensinTM at least 15 days prior to sampling

for control observations.

60
Diets

 

Diets are given in Tables 3 and 4. The only difference was the
added RumensinTM to the supplement.
Sampling

A11 sampling was done 4 hours post-feeding and samples were
handled as discussed previously.
Analysis

Data were analyzed using a paired T-test. Observations from the
no-monensin treatment was compared to monensin-supplemented
observations and mean differences were tested. There were no

significant differences due to sex so all observations were pooled.

61
Results
Study 1 In Vitro Addition of Monensin to Duodenal Mucosal Biopsies

Monensin addition to incubated biopsies resulted in increased
total oxygen consumption and increased ouabain-sensitive respiration
(Na+/K*-ATPase). There was no effect on Na+/K+-ATPase-
independent reSpiration. Means and significance levels are presented
in Table 5.

Total 02 consumption of the duodenal mucosal biopsies without
monensin supplementation (control) was 5.59 nmol 02/mg DM/min. 02
consumption increased with increasing monensin concentration. Control
02 consumption rates differed significantly from all monensin
concentrations examined (P<.01). One ppm monensin increased 02
consumption to 6.40 nmol 02/mg DM/min, 5 ppm monensin increased
consumption to 6.79 nmol 02/mg DM/min, and 10 ppm monensin increased
consumption to 8.18 nmol Oz/mg DM/min. When 1 ppm monensin was
compared to 5 and 10 ppm monensin, 02 consumption was fbund to be
significantly different (P<.05). The response in total 02
consumption determined at 5 ppm also differed significantly from that
seen at 10 ppm monensin.

Ouabain-sensitive, Na+/K+-ATPase-dependent respiration rates
increased significantly (P<.01) with increasing concentrations of
monensin in incubation media. Control 02 consumption rates were
fbund to be 2.76 nmol 02/mg DM/min. This rate increased to 3.77 nmol
02/mg DM/min with addition of 1 ppm monensin, 4.15 nmol Ozlmg
DM/min with 5 ppm monensin and 4.9 nmol 02/mg DM/min with 10 ppm

monensin. One ppm monensin differed significantly from 5 and 10 ppm

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62

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mm cam: mm com: um new: newspemte
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mmmah<i+x\+mz

 

 

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63
monensin (P<.05) and 5 ppm was significantly lower than 10 ppm
(P<.lO).

Monensin addition did not affect Na+/K+-ATPase-independent
respiration regardless of concentration. Control respiration rates
were 2.83 nmol OZ/mg DM/min, while respiration rates from monensin
treated biopsies ranged from 2.64 nmol 02/mg DM/min to 3.36 nmol
Oz/mg DM/min.

Study 2: The Effect of Monensin Supplementation On Duodenal Mucosal
Mal/KT-ATPase Activity

Monensin addition to the diets of beef cattle significantly
increased total 02 consumption (P<.Ol) and NaT/K+-ATPase-
dependent respiration (P<.01). There was no effect on
Na+/K+-ATPase-independent respiration although there was a
numerical increase (2.68 nmol Oz/mg DM/min versus 2.85 nmol
02/mg OM/min). Means are presented in Table 6. .

Control total 02 consumption was 5.85 nmol Ozlmg DM/min and
monensin supplementation increased this to 7.03 nmol 02/mg DM/min.
Ouabain-sensitive respiration increased from 3.14 nmol Oz/mg DM/min
to 4.15 nmol Ozlmg OM/min, an increase of 32%. The percent
inhibition by ouabain increased from 53.6% to 58%.

There was no significant differences between heifers and steers in

either their control 02 consumption rates or monensin supplemented

rates.

64

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.mmwmaown em “commend; mcemzm

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co_uecwammc cowumtwqmme cowuqeamcou mo
uemccmaouee- uemuemqmv- ~euo»

 

omeae<-+¥\+ez

 

 

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65
Discussion

Total oxygen consumption measurements for the control treatment in
both studies (5.59 and 5.85 nmol Oz/mg/min) are higher than those
reported by McBride (1984) for lactating and dry, pregnant dairy
cattle. His values ranged from 3.89 1 .41 nmol Ozlmg DM/min fOr cows
in week 5 of lactation to a high of 4.92 i_.l7 nmol Ozlmg DM/min at
week 22-25. They are also higher than the 4.65 nmol OZ/mg DM/min
reported by Webster and White (1973) fbr the entire gastrointestinal
tract of the sheep. However, these data are in line with that reported
by McBride (1984) fbr sheep feed different levels of digestible energy.
In this study his mucosal 02 consumption (nmol Ozlmg DM/min) values
ranged from 5.21 for starved sheep to 6.07 for sheep fed a higher
digestible energy diet. Estimates of ouabain-sensitive respiration
(Na+/K+-ATPase-dependent respiration) and ouabain-insensitive
respiration (NaT/KT-ATPase independent) also agree with McBride
(1984). This is likely to be related to energy intake. Like the sheep
in his energy intake study, the cattle in this study were fed a high
energy diet.

When monensin was added to the media incubating biopsies the rate
of oxygen consumption, both total and NaT/KT-ATPase-dependent
respiration increased significantly. Total mucosal oxygen consumption
increased linearly (R2=.94) with increasing monensin concentration
and Na+/K+-ATPase-dependent respiration increased linearly
(R2=.85). The increase in total 02 consumption is due primarily to
a stimulation of the Na+/K+-ATPase, since Nat/K+-ATPase-

independent respiration rates did not change (although at 10 ppm

bb
monensin, 3.36 nmol 02/mg DM/min was consumed which is a 15% increase
over that found in control incubations).

The significant increase in NaT/KT-ATPase-dependent
respiration is likely to be due to the action of monensin on the flux
of sodium ions into the enterocyte. Monensin has been shown to
increase sodium concentrations intracellularly in 313 cells (Smith and
Rozengurt, 1978; Austic and Smith, 1980). Both studies with 3T3 cells
used concentrations of'monensin similar to those used in this study.
One, 5 and 10 ppm (1, 5 and 10 ug/ml) were used in these studies and
they used 1-20 ug/ml monensin.

The magnitude of the increase in pump activity seen with monensin
is different between studies. In the 3T3 cells approximately 3 ug/ml
monensin provided maximal stimulation. This stimulation was a 6 fold
increase in activity. The present data indicate a 1.7 fold increase in
Na+/K+-ATPase-dependent respiration from O to 10 ug/ml. This is
probably due to the difference in tissue type but may also reflect a
difference in the amount of monensin reaching the cell. Biopsies would
not be as likely to have a unifbrm distribution of cells like a 3T3
cell culture experiment.

Addition of monensin to steers and heifers on feedlot diets
significantly increased total mucosal 02 consumption (P<.Ol) and
Na+/K+-ATPase-dependent respiration rates (P<.01). Total mucosal
Oz consumption increased from 5.85 nmol 02/mg DM/min in duodenal
biopsies from steers not fed monensin to 7.03 nmol 02/mg DM/min in
duodenal biopsies from steers supplemented with monensin. Again the

increase in total mucosal 02 consumption is likely to be due to the

67
significant increase in NaT/K+-ATPase-dependent respiration. The
activity of this pump, as measured by oxygen consumption, increased
1.32 fbld from 3.14 nmol OZ/mg DM/min to 4.15 nmol Oz/mg DM/min.

The pr0portion of respiration that was inhibited by ouabain
increased from 53.6% to 59% reflecting an increase in Na+/K+-ATPase
activity. This is also in keeping with data of other workers who
indicate that monensin stimulates sodium uptake into the cytosol of the
cell (Smith and Rosengurt, 1978, Austic and Smith, 1980). This
stimulation is independent of membrane potential and doesn't interfere
with the electrochemical gradient (Smith and Austic, 1980; Bihler et
a1, 1985). The increased intracellular sodium stimulates the
Na+/K+-ATPase pump. Bihler et a1. (1985) also noted that monensin
decreased intracellular ATP concentrations in avian erythrocytes.

The amount of'Oz utilized/mg of dry intestinal tissue was 7.03
nmol Ozlmg/min. This value is a 10% increase over that seen by
McBride (1984) with sheep fed high energy diets and is within the range
(6.55-7.18 nmol 02/mg/min) reported by Levin and Syme (1975) and
Liberman et a1. (1979). To what degree a 20% increase in 02
utilization by the gastrointestinal tract would effect an animal's
maintenance requirements would depend upon the degree to which other
monensin effects (e.g. increased propionate production) contribute to
that requirement.

The increase in Na+/K+-ATPase activity may also have other
implications in the gastrointestinal tract. The Nat/Ki-ATPase pump
has been shown to be associated with coupled transport of glucose,
amino acids, sodium and chloride. An alteration in NaT/K+-ATPase

activity by 32% might significantly affect transport of these

nutrients.

In Vivo: Net Nutrient Absorption by the Gastrointestinal Tract of

Growing Beef Steers

68

Introduction

The previously reported in vitro studies indicated a 20% increase
in total mucosal oxygen consumption with monensin feeding and a 32%
increase in the activity of the Na+/K+-ATPase pump in mucosal
biopsies. Since the NaT/Kl-ATPase pump was stimulated in beef
steers and heifers fed monensin as RumensinTM, and since the
Na+/K+-ATPase pump is an important constituent of glucose (Crane,
1961), amino acid (Crane, 1977), and mineral (Bihler and Cybulsky,
1973) absorption in the small intestine a study was conducted to
evaluate whether monensin supplementation altered net absorption of
nutrients across the intestinal tract.

The hypothesis of this study was that monensin had no effect upon
glucose, alpha amino nitrogen, lactate and mineral absorption across

the intestinal tract as measured by appearance in the portal vein.

Materials and Methods

Animals

Four steers averaging approximately 363 kg in weight were used in
a switchback design to evaluate the effect of monensin feeding upon net
nutrient absorption from the gastrointestinal tract. All of the steers
were Simmental crossbreeds. The steers were housed in the metabolism
room in individual pens measuring 1.8 x 2.4 m. They had free access to
water and there was no bedding as the pens have slotted floors. Steers
were either haltered or held in stanchions when cannulated to prevent
licking at catheters.
9.1.9.2

Diet composition is given in Table 7. On a dry matter basis it

69

70

Table 7. Diet Composition for Net Absorption Studies

 

 

 

Ingredient % OM
High Moisture Ground Ear Corn 75
Ground Hay 21
Supplement1 4

 

1supplement contained soybean meal (95%), trace

mineral salt (2%), limestone (1%), selenium 90 (2.4%)

and Vitamins A, D and E. RumensinTM was added at

200 mg/lb.

ll

consisted of 75% high moisture whole shelled corn, 21% ground hay and
4% supplement. Monensin was added as RumensinTM in the supplement
at a level of 300 mg/head/day when the animals received the monensin
treatment. The daily ration was fed 12 times/day via automatic feeders
positioned over the pens. The diet was fbrmulated to meet NRC (1984)
requirements for steers gaining 0.68 kg/day.
Catheterization

Catheters were placed in the portal vein, mesenteric vein (2),
Ltwo catheters were placed in mesenteric veins to allow infusion of dye
in case one catheter closedJ and iliac artery using methods developed
by McGillard and Thorp (1971) and Huntington (l982).‘ Portal vein
catheters were made of teflon (Essex Group, Magnet Wire and Insulation
Division, Ft. Wayne, Indiana) and were encased in silastic tubing.
Mesenteric vein and iliac artery catheters were made of'tygon tubing
and were treated repeatedly with TDMAC-heparin (Polysciences Inc.,
Warrington, PA), to help prevent clotting, befbre sterilization. All
catheters were washed and gas sterilized prior to surgery.
Exteriorization of portal and mesenteric catheters was done along the
spine. Iliac artery catheters were exteriorized above the incision
site. Catheters were then placed in patches which were cemented to the
hair with tag cement. All catheters were filled with physiological
sterile saline containing 200 units of heparin/ml and 1% penicillin

capped and left alone until collection or until an animal had disturbed

them.
Protocol

Prior to surgery each steer was adjusted to the experimental diet

72

and the metabolism room for several days. After surgery animals were
returned to the metabolism room and brought back up on full feed
gradually. At this time the animals which were to receive monensin
were supplemented with RumensinTM and adjusted to 300 mg/hd/day.
Animals were treated with antibiotics for the first 7 days postsurgery.
Generally it was 10-15 days before steers were on full feed. At this
time a blood flow measurement was made to validate correct placement of
the portal catheter. This resulted in 3 steers being removed from the
study. Two of the three had nonphysiological blood flows (2940 L/hr,
1511.25 L/hr), i.e., too rapid, suggesting incorrect placement of the
portal catheter. The other had extremely low blood flows (189.7 L/hr)
again suggesting improper catheter placement. These steers were
replaced with newly cannulated steers whose blood flows were
physiological.

After the steers had been on full feed fbr at least 5 consecutive
days blood collections were initiated. .
Blood Flow Measurements

Blood flow through the portal vein was measured by dye dilution
techniques using p-aminohippuric acid (PAH) originally used by Roe et
a1 (1966) and modified by Katz and Bergman (1969) and Huntington
(1982). Using a Harvard infusion pump, a bolus of PAH was infused far
15 minutes. This bolus was a solution of PAH containing 132.6 mg/ml
infused at a rate of 1.5 ml/min. After the 15 minutes of infusion the
pump was turned down to deliver at a rate of .754 ml/min. This is
equivalent to an infusion rate of 6 gm PAH/hr. Blood samples from the

iliac artery and portal vein catheters were withdrawn simultaneously

73

over a 2 minute period. The slow withdrawal period was to help insure
adequate mixing of dye. Each animal was sampled in 4 hour blocks for a
24 hour period of time. This was done over several days. There was a
span of at least 8 hours between infusions.
Sample handling

Blood samples were collected in Sarstedt tubes containing lithium
heparin as an anticoagulant and were used for determination of plasma
minerals, total lipid, a-amino nitrogen and blood urea nitrogen. Blood
samples that were to be analyzed for glucose were collected in tubes
containing sodium fluoride. Blood samples collected in both types of
tubes were placed on ice immediately and centrifuged as soon as
possible (about 30 min.). An initial 0.5 ml volume of whole blood was
placed in 5.5 ml ice cold 10% trichloroacetic acid (TCA), vortexed,
centrifuged and placed in a cooler (4°C) fbr subsequent analysis for
PAH. A blood sample was also taken for packed cell volume analysis for
each infusion period. Twenty ul of whole blood was added to a
hemolysis tube with .99 ml of a sodium azide solution (1 gm/L) and
frozen immediately far later lactate analysis. Blood samples for
glucose and lactate analysis were taken every hour and for PAH every
half hour. After centrifuging, plasma samples were frozen for later
analyses.
Assays

LT-lactate

L+-1actate was analyzed using a Kontron 640 Lactate Analyzer
(courtesy Dr. Wayne Van Huss of the Human Performance Lab). One

hundred pl of hemolyzed whole blood in sodium azide was injected into

74
the analyzer and mmol/L lactate was read off the screen. The
measurement of lactate is achieved by monitoring the concentration of
hexacyanoferrate II fermed during the irreversible oxidation of lactate
to pyruvate in the presence of cytochrome b2. For each lactate
molecule oxidized, two electrons are collected on the platinum
electrode creating a measurable current. The reactions are:
i. Lactate + 2LFe(CN)6_)'3 EZE_9§ pyruvate + 2 LFe(CN)6]'4
ii. 2LFe(CN)5J'4 _"_E__, 2 LFe(CN)5_|'3+2e'

This procedure is specific fbr L+-1actate.

Glucose

Plasma glucose concentrations were measured using the glucose
(Trinder) kit (Sigma Diagnostics #315). The reactions involved are the
oxidation of glucose to gluconic acid and hydrogen peroxide and the
subsequent reaction of the hydrogen peroxide with 4-amino antipyrene
and p-hydroxybenzone sulfbnate. This forms a quinoneimine dye which is
read spectrophotometrically at 505 nm. Absorbance readings are then
regressed along a standard curve of glucose concentrations fbr
quantitation.
Blood Urea Nitrogep

Blood urea nitrogen (BUN) concentration was measured using a Sigma
Diagnostics kit ( 535). In this procedure urea reacts with diacetyl
monoxime to form a pink chromogen. The concentration of this color
production is read spectrophotometrically at 535 nm and BUN

concentration is determined from a urea nitrogen standard curve.

75
Alpha amino nitrogen

Alpha amino nitrogen (AAN) was analyzed by using an adaptation of
a procedure by Palmer and Peters (1967). Samples were deproteinized
with 10% trichloroacetic acid and 0.2 ml of filtrate used fbr analysis.
To the protein free filtrate 1.6 m1 of .OSM sodium borate and 0.2 m1 of
0.25% trinitrobenzene sulfbnic acid (TNBS) was added. The mixture was
incubated at 37°C fbr 20 minutes. 2 m1 of 1N HCl were added and the
tube vortexed. The sample was read on a spectrophotometer at 420 nm.
Citrulline was used to make a standard curve which was then used fbr
quantitation of'AAN.

The basis for this procedure is the production of a TNBS-amino
acid complex at pH 9.2. This complex is unstable and upon
acidification (1N HCl) farms a stable yellow trinitrophenol-amino acid
derivative.

Total Lipid

Total lipid was measured by using an adaptation of Hara and Radins
(1978) procedure. Two ml of plasma were added to a 20 x 150 mm screw
cap tube with a teflon lined cap. Nine ml of a 3:2 hexane:isopropanol
mixture were added and the tube vortexed 30 seconds. The tubes were
then allowed to separate and 7 ml of’a 6.7% NaSO4 solution was added.
The tubes were vortexed 30 seconds again and allowed to separate. The
supernatant was pipetted to a 20 m1 test tube and the meniscus washed
with 4 m1 of a 7:2 mixture of hexane:iSOpr0panol. The solvent was then
evaporated in a heated sand bath under nitrogen until 1-2 ml remained.
This was transferred to a weighed 12 x 75 mm test tube and the 20 m1

tube was washed with 2 ml of a 7:2 hexane:150propanol mixture. The

76

solvent was then evaporated completely from the 12 x 75 mm test tube in
a heated sandbath under nitrogen. The tube was then reweighed and
total lipid was calculated from the difference in weights.
p-Aminohippuric acid

p-Aminohippuric acid concentrations in the whole blood of steers
were analyzed using the procedure outlined by Katz and Bergman (1969)
and Huntington (1982). A protein free supernatant was prepared
immediately after blood collection (see Blood Flow Measurements). One
ml of this supernatant was added to a disposable 13 x 100 mm Kimax
tube. To this tube 0.2 m1 of 1.2 N HCl, 0.1 m1 of 100 mg/dl NaNOZ,
0.1 ml of 500 mg % ammonium sulfamate, 0.1 ml of 100 mg/dl
(N-(l-napthyl) ethylenediamine dihydrochloride were added. After 10-15
minutes the absorbance was read at 540 nm on using a spectrophotometer.
Standard curves were made using the infusate and were used fur
quantitation of PAH concentrations.
Mineral AnaLysis

Chloride

Plasma chloride was analyzed using a Digital Chloridometer
(Buchler model 4-2500). The principle of analysis is the generation of
silver ions at a silver anode. These ions are complexed with chloride
ions present in the sample and are precipitated. When all available
chloride in the sample has reacted, the free silver ion generates an
increase in current which is sensed at the indicator electrodes. At
this point the action stops and a reading in mEq/L is generated.

Phosphorus

Phosphorus was analyzed using the Gomori modification of the

Fiske-SubbaRow (1946) inorganic phosphate determination.

77

W

The Whetter and Ullrey (1978) variation of the AOAC procedure far
the spectrofluorometric determination of selenium was used to determine
plasma selenium concentrations. The principle is the farmation of a
selenium-diamino-napthalene complex that is light sensitive, soluble in
cyclohexane and fluorescent. The fluorecence is regressed against a
standard curve fbr quantitation of plasma selenium.

Magnesium, Calcium, Copper and Zinc

Plasma magnesium, calcium, copper and zinc concentrations were
determined using a model IL 951 (Wilmington, MA) atomic absorption (AA)
spectrophotometer.

Plasma samples were deproteinized in 12.5% trichloroacetic acid
and then diluted to appropriate concentrations and read. Wavelengths
(nm) used were magnesium, 285.2; calcium, 427.7; copper, 342.7; and
zinc, 313.9. The AA regresses readings against a standard curve and
gives actual concentration readings.

Potassium and Sodium

Potassium and sodium concentrations in plasma was determined using
a Model IL 951 atomic emission spectrophotometer (Wilmington, MA).

This procedure differs from atomic absorption in that only the flame
and no lamps are used. Concentrations are calculated from a
standard curve. Wave lengths (nm) used were: sodium, 589 and
potassium, 766.5

Statistical Analysis

The original design was a switchback with each animal serving as

its own control. Because of problems with intake, heat, possible

78
infections and eventual catheter malfunction. The original design of
this study could not be carried out. Measurements with and without
monensin supplementation were to have been made for each animal,
however. The eventual data set included 2 steers on each treatment.
This became a problem to analyze and detect differences. Ultimately I
settled far little power and analyzed the study using a split-plot
analysis with one degree of freedom for treatment. In the subplot,
time within treatment was never significantly different so it was
dropped and its degrees of freedom added to residual. Time was also
not different. This is not surprising because everything was done to
minimize time effects. Feeding 12 times a day reduces some of the
variation, if not all, in digesta flow to the small intestine (Weber
and Rumpler, personal communication). Sampling every half hour also
minimized any time effects in blood flow.

The general linear models (GLM) procedure of the SAS (SAS
Institute, N.C.) computer package was used to generate least squares
means and test for treatment differences.

Calculations

Blood flow was calculated using the fallowing equation developed

by Katz and Bergman (1969):

= I
FPV CPV-CA

Where: I = infusion rate of PAH (mg/min)
cpv = concentration of PAH in the portal blood (mg/m1)
CA = concentration of PAH in arterial blood (mg/m1)

va = flow through the portal vein (ml/min or L/hr)

79

Net absorption was calculated using the equations of Bergman et a1
(1974).
Net portal absorption (NPA) = Fp(CP-CA)

Where: Fp = portal plasma flow (L/hr)
CP = metabolite concentration in portal blood (units/m1)
CA = metabolite concentration in arterial blood

(units/ml).

Results

In this study, there was no effect of monensin on dry matter
intake. Control steers consumed approximately 7.86 kg of DM/day while
monensin fed steers consumed 7.89 kg DM/day. There was also no effect
of monensin on animal performance. All steers averaged 0.70 kg
gain/head/day while cannulated.

Arterial and Portal Blood Concentration Differences

Minerals

Mean arterial and portal blood concentrations of calcium and
phosphorus are given in Table 8. Monensin supplementation
significantly increased arterial calcium concentrations (P<.08) from
10.48 mg/dl in control animals to 11.28 mg/dl in monensin supplemented
steers. Portal blood calcium concentrations were numerically greater
fbr monensin-fed steers (11.35 mg/dl) than far control animals (10.85
mg/dl) but this was not significantly different (P>.10). Arterial
blood phosphorus concentrations tended to be lower fbr cattle fed
monensin (6.37 mg/dl) than for steers fed the control diet (7.43
mg/dl). This was not fbund to be significantly different (P>.10).

Portal phOSphorus concentrations were significantly different,
however (P<.01). Control portal phosphorus concentrations (7.72 mg/dl)
were higher than those determined in monensin fed cattle (6.70 mg/dl).

There were no significant differences (P>.10) in mean arterial or
mean portal concentrations of magnesium, copper, zinc or selenium
(Table 9). Copper and zinc concentrations did tend to be higher for

monensin supplemented steers but the large amount of variation,

80

81

Table 8. Mean Arterial and Portal Blood Concentrations
of Calcium and Ph05phorus from Control and Monensin
Supplemented Steers1

 

 

 

 

 

 

 

 

Control Monensin
A p A P
Ca (mg/d1) 10.482 10.35 11.282 11.35
2.30 :.34 1.30 :.34
P (mg/d1) 7.43 7.723 6.37 6.703
1.39 2.129 2.39 i.129

 

1Means are 24 samples analyzed in duplicate.
2Means with the same superscript differ P<.08.
3Means with the same superscript differ P<.Ol.

82

Table 9. Mean Arterial and Portal Blood Concentrations
of Magnesium, Capper, Zinc and Selenium from Control and
Monensin Supplemented Steersl

 

 

 

 

Control Monensin
A P A P

Mg (mg/d1) 2.41 2.34 2.32 2.78
2.22 2.21 2.22 2.21

Cu (ug/di) 121.26 123.87 145.45 140.83
227.08 223.31 227.08 223.31

Zn lug/m1) 121.74 113.17 138.47 125.72
210.59 241.82 210.59 241.82

Se (ug/ml) .091 .091 .086 .089
2.007 2.006 2.007 2.006

 

 

 

1Means represent 24 observations analyzed in duplicate.

83

approximately l8% for copper and 8% for zinc, prevented the detection
of any possible differences.

Arterial concentrations of sodium, potassium and chloride showed
no significant differences (P>.l0) when comparing control and monensin
supplemented animals (Table l0). Sodium concentrations tended to
increase when monensin was supplemented (2l8.4l mg/dl vs. 223.95
mg/dl). Potassium concentrations also tended to increase with monensin
supplementation. Portal blood concentrations of both sodium and
potassium were significantly increased (P<.lO) in monensin fed animals.
Mean sodium concentrations in portal blood for control animals were
220.46 mg/dl and 232.79 mg/dl in the supplemented steers. Potassium
concentrations increased from 17.36 mg/dl to 18.49 mg/dl with monensin
supplementation. Portal chloride concentrations were not different.

It should be noted that all of the sodium values appear to be slightly
lower than reported normal values. Normal sodium levels in bovine
whole blood range from 260-280 mg/dl which is equivalent to 113-121
mEq/L. .

Lactate, Glucose and Totalgipid

L-lactate, glucose and total lipid concentrations in the arterial
and portal blood were not found to be significantly different between
the control and monensin treated animals. Lactate concentrations in
both arterial and portal blood tended to be lower in monensin
supplemented steers than control steers (Table ll). In contrast,
glucose concentrations showed the opposite trend. Both arterial and
portal glucose concentrations appeared to increase in monensin fed

steers.

Table l0.

84

Mean Arterial and Portal Blood Concentration of Sodium,

Potassium and Chloride from Control and Monensin-Supplemented
SteersLZ’3 (mg/d1)

 

 

Control

A

Na 218.41 (95.0)
113.06 (5.68)

K 16.98 (4.35)

21.32 (.34)
Cl 339.32 (96.95)
22.48 (.71)

P

220.462 (95.9)
24.24 (1.84)

17.362 (4.44)

$1.02

330.47 (96.42)

12.56

(.26)

(.73)

Monensin

A

P

223.95 (97.42) 232.792 (101.26)

213.06 (5.68)

17.43 (4.46)
21.32 (.34)

338.77 (96.79)
22.48 (.71)

24.24

18.492
21.02

328.65
$2.56

(1.84)

(4.73)
(.26)

(93.90)
(.73)

 

- 1Means represent 24 observations.

2Means in a row with similar superscripts differ P<.10.

3Values in parentheses are mEq/L.

85

Table ll. Mean Arterial and Portal Blood Concentrations
of L-Lactate. Glucose. and Total Lipids in Control and
Monensin Supplemented Steers ‘

 

 

 

 

 

 

Control Monensin
A P A P
L-Lactate (mM)1 .936 .961 .906 .917
2.11 2.15 2.11 2.15
Glucose (mM)1 4.02 3.98 4.34 4.28
2.14 2.24 2.14 2.24
Total Lipid (mg/dl)2 233.61 241.67 197.0 212.43
234.4 230.81 234.4 230.81

 

1Means represent 48 observations analyzed in duplicate.
2Means represent 24 observations analyzed in duplicate.

86

Alpha Amino Nitrogen and Blood Urea Nitrogen

Supplementation of monensin did not significantly alter a-amino
nitrogen and blood urea nitrogen concentrations in either arterial or
portal blood. In both arterial and portal blood, a-amino nitrogen
concentrations tended to increase in steers supplemented with monensin
while blood urea nitrogen concentrations appeared to decline when
monensin was fed (Table 12).
Blood Flow Estimates

Estimates of portal blood flow rates were not significantly
different with addition of monensin (Table 13). Control steers
averaged 623.93 L/hr while those steers supplemented with monensin had
blood flow rates of 607.18 L/hr. The standard error of the differences
for these estimates is 12-13% of flow values which is a reasonably low
error percentage for studies of this type.
Portal-Arterial and Net Absorption Differences

Portal-arterial differences can be obtained by two methods. One
method, and the way that most workers use, is to take the mean arterial
concentration and subtract it from the mean portal concentration of the
metabolite of interest. Using this method a representative difference
for a time period is calculated. This number is then multiplied by a
mean blood flow value and net absorption rates may be calculated.
Other workers in the field use a mean P-A difference and a mean net
absorption value calculated from a concentration difference and its
blood flow rate. By this method statistics may be run and comparisons
can be made between treatments. Using either method the values
obtained are usually similar although not identical. In this study

mean P-A differences were generated and examined statistically.

87

Table 12. Mean Arterial and Portal Blood Concentrations
of Alpha Amino Nitrogen (AAN) and Blood Urea Nitrogen
(BUN) from Control and Monensin Supplemented Steers1

 

 

 

 

 

Control Monensin
__A__ P _JL_ P
A-AN (mM) .783 1.083 .803 1.175
1.015 .1.02 1.015 $.02
BUN (mM) 3.30 3.29 3.14 2.92
$.04 $.01 1.04 1.01

 

 

1Means represent 48 observations in duplicate.

88

Table 13. Blood Flow Estimates (L/hr) from Control
and Monensin Supplemented Steers1

 

 

Control Monensin SED

 

623.93 607.18 78.70

 

1Means represent 96 observations.

89
Net absorption rates were calculated using a mean blood flow estimate
multiplied by a mean P-A difference. This means that a true standard
error of the net absorption estimate cannot be calculated.

Portal-arterial calcium concentration differences were
significantly different with monensin supplementation (Table 14). The
difference between portal and arterial concentrations was smaller with
monensin reflecting the increased arterial calcium concentrations
reported earlier (Table 8). When putting the differences on a blood
flow basis, net absorption rates were 2.3 g calcium/hr for control and
.425 g calcium/hr f0r monensin supplemented cattle.

There were no differences (P>.10) in P-A differences for
phosphorus despite the significant decrease in portal phosphorus
concentrations (Table 8). Net absorption rates were 1.81 g/hr f0r
control steers and 2.00 mg/hr for monensin supplemented steers.

Table 15 shows P-A differences and net absorption rates for
magnesium, copper, zinc and selenium. Magnesium P-A differences were
significantly different (P<.02). This indicates net absorption of
magnesium through the gastrointestinal tract into the portal blood with
monensin treatment. Net absorption rates were negative for control
steers indicating no net output of magnesium into the portal blood.
Rates were -.392 g/hr for control and .243 g/hr for monensin
supplemented animals. Copper P-A differences were not different
(P>.10) nor were zinc and selenium P-A differences found to be
different (P>.10).

Portal-arterial differences were not different (P>.10) between
treatments for sodium, potassium or chloride (Table 16). Net

absorption rates are also given. There was a tendency to show

90
Table 14. Mean Portal-Arterial (P-A) Differences and Net
Absorption Rates (NA) in Control and Monensin Supplemented

Steers: Calcium and Phosphorus

 

 

Control Monensin ‘SEQ
Calcium
P-A (mg/dl) .371 .071 .171
NA (g/hr) 2.3 .425
Phosphorus
P-A (mg/dl) .29 .33 1.04
NA (g/hr) 1.81 2.00

 

1Means with the same superscript are different P<.08.

91

Table 15. Mean Portal-Arterial (P-A) Differences and
Net Absorption Rates (NA) in Control and Monensin
Supplemented Steers: Magnesium, Copper Zinc and Selenium

 

 

Control Monensin _§§Q_
Magnesium
P-A (mg/d1) -.0631 .041 .0237
NA (glhr) -.392 .243
Copper
P-A (ug/dl) 2.61 -4.62 3.83
NA (mg/hr) 16.28 -28.05
Zinc
P-A (ug/dl) -8.58 35.18 34.94
NA (mg/hr) -53.53 213.61
Selenium
P-A (ug/dl) -.001 .003 .0013
NA (pg/hr) -6.23 18.21

 

1Means with the same superscript are significantly

different P<.02.

Table 16. Mean Portal - Arterial (PA) Differences and Net
Absorption Rates (NA) in Control and Monensin Supplemented

 

 

Steers: Sodium, Potassium and Chloride
Control Monensin gag;
Sodium
P—A (mg/d1) 2.05 8.84 14.28
NA (g/hr) 12.79 53.67
Potassium
P-A (mg/d1) .38 1.06 1.905
NA (g/hr) 2.37 6.44
Chloride
P-A (mg/d1) - 8.93 -10.12 1.25
NA (9/hr) -55.72 -61.45

 

93
increased sodium and potassium absorption into the portal blood for
monensin treated steers (Table 16). Control steers had net absorption
rates of 12.79 g Na/hr and 2.37 g K/hr, while monensin supplemented
steers had absorption rates of 53.567 9 Na/hr and 6.44 g K/hr.
Chloride absorption values were similar for both treatments (P>.10).

P-A differences in lactate reflect the tendency f0r decreased
lactate concentrations in arterial and portal blood with monensin
supplementation (Table 17). Control steers had net absorption rates of
15.60 m/hr while monensin supplemented animals had net absorption rates
of 6.68 m/hr. Glucose P-A differences and net absorption rates were
lower f0r animals supplemented with monensin. These differences were
not significantly different (P>.10). Total lipid P-A differences were
also not different (P>.10).

There were no significant differences (P>.10) in P-A differences
in alpha amino nitrogen or blood urea nitrogen concentrations.
Numerically there appears to be a stimulation of alpha amino nitrogen
transport into the portal vein. Absorption rates were 187.18 m/hr for
control steers and 226.48 m/hr f0r monensin supplemented animals.
Blood urea nitrogen showed the opposite trend. In this case there was
a tendency fer blood urea nitrogen absorption rates to be lower in
those animals receiving monensin (-133.57 m/hr vs. -6.24 m/hr for

control).

94
Table 17. Mean Portal-Arterial (P-A) Differences and Net
Absorption Rates (NA) Rates in Control and Monensin

Supplemented Steers: Lactate, Glucose and Total Lipids

 

 

Control Monensin SEQ
Lactate
P-A (mM) .025 .001 .074
NA (mM/hr) 15.60 6.68
Glucose
P-A (mM) -.04 -.06 .114
NA (mM/hr) -24.95 -36.43
Total Lipid
P-A (mg/d1) 18.06 15.43 16.87

NA (g/hr) 112.68 93.69

 

'95
Table 18. Mean Portal-Arterial (P-A) Differences and Net
Absorption (NA) in Control and Monensin Supplemented

Steers: Alpha Amino Nitrogen (AAN) and Blood Urea

Nitrogen (BUN).

 

 

Control Monensin ‘ SEQ
A-AN
P-A (mM) .30 .37 .235
NA (mM/hr) 187.18 226.48
BUN
P-A (mM) -.01 -.22 .963

NA (mM/hr) -6.24 -133.57

 

Discussion

It is truly an understatement that this study was without any
difficulty. It was originally planned as a disappearance (of digesta)
appearance (of metabolites in the portal blood) study but this became
impossible. Early attempts at catheterization of the portal vein were
not successful in maintaining the catheters f0r periods longer than 30
days. The problem seemed to be the inability to suture the catheter to
the vein to hold it in position. Communications with Dr. G. Huntington
(U.S.D.A., Beltsville, MD) indicated that he could not pull a portal
catheter out easily, I could. With such a short catheter patency, it
became essential that the steers be placed on feed as quickly as
possible. The cannulation of the duodenum, although always successful,
appeared to slow the recovery of appetite. These animals would come
back on feed as usual but then back off. It was then decided to forgo
disappearance and concentrate on measuring net portal appearance.

Initially the right cartid artery was exteriorized for later
catherization f0r blood flow studies. Although this was successful,
healing was not rapid (probably because of stanchions). However after
‘a carotid artery burst, I opted to switch to catheterization of the
iliac artery. A catheter was placed in the iliac artery during
surgery. These preparations healed quickly and did not cause any
further problems.

A total of 8 steers were catheterized. Three of the steers which
were not used, were found to have non-physiological blood flow
estimates indicating incorrect catheter placement and another steer

died after uncontrollable carotid bleeding. Four steers were

96

97
subsequently used in the study.

Monensin supplementation at 300 mg/head/day had several effects on
mineral composition of blood plasma and net absorption. Arterial
calcium concentrations significantly increased (P<.08) from 10.48 mg/dl
to 11.28 mg/dl. It did not significantly (P>.10) increase portal
concentrations although they did increase numerically. Despite an
increase in arterial calcium concentration, net absorption of calcium
was not increased (Table 14). Instead it was decreased from 2.3 g/hr
to .425 mg/hr.

It is important to view these findings in their entirety.

Arterial and venous concentrations and their differences may be
interesting and significant, but the most important criteria must be
net absorption. For example there was no difference of'monensin on
total blood lipid concentrations (Table 17) with added monensin. There
was also no significant differences in the P-A difference of lipids,
however when blood flow rates are applied to the P-A difference it was
determined that net absorption of lipids was over 2 kg/day. This is
highly unlikely. The calcium data would indicate that net absorption
from the rumen and intestine is decreased and the animal retained more
calcium.

Monensin also decreased (P<.01) portal concentration of phosphorus
from 7.72 to 6.70 mg/dl. It had no affect on arterial phosphorus
concentration (7.43 mg/dl-control and 6.37 mg/dl fer supplemented
steers). P-A differnces of phosphorus were not different and net

absorption (NA) rates were also unaffected (Table 14).

98

There was no significant effects of monensin supplementation on
portal blood or arterial concentrations of magnesium, copper, zinc or
calcium. Concentration of copper and zinc showed numerical increases
with monensin supplementation. This agrees with data reported by
Starnes et a1. (1984) who also reported elevated c0pper and zinc levels
in jugular blood from beef steers supplemented with monensin. Starnes
et a1. (1984) offered no explanation as to why serum levels of zinc and
c0pper were higher in animals receiving ion0phores. It would be
attractive to speculate that the increase in plasma zinc and c0pper
levels is an absorption effect, however, the data in this study do not
support such an effect (Table 15).

Magnesium concentratfions were not significantly altered by
monensin supplementation but the P-A difference of magnesium was
significantly elevated in monensin supplemented animals (Table 15).
This resulted in net absorption estimates of -39.3 g/hr in control and
24.73 g/hr in supplemented animals. This clearly demonstrates that
magnesium absorption from the rumen was altered by monensin. This
magnesium effect has been speculated by Greene et al. (1985) and
Starnes et a1. (1984). These researchers demonstrated an apparent net
increase in magnesium retention in beef cattle and sheep fed monensin.
This maybe due to a stimulation by monensin of the Na+/K+-ATPse
activity in the rumen epithelium. Martens (1983) demonstrated that
magnesium uptake was inhibited with ouabain administration. This
stimulation of magnesium uptake may have important implications in

prevention of grass tetany.

99

Portal concentrations of sodium and potassium were increased with
monensin supplementation (Table 10). but P-A differences were not
different. Net absorption rates indicated a numeric increase in sodium
(1.28 g/hr versus 5.37 g/hr) and potassium (2.37 g/hr versus 6.44 g/hr)
with monensin supplementation. Elevated portal concentrations of both
minerals may be a result of an increase in either ruminal absorption or
an increase in intestinal absorption. Both mechanisms of absorption
would be stimulated by the presence of monensin because both are sodium
dependent processes (Birge et a1, 1974; Nasserman, 1981). It is not
surprising to find no change in arterial concentrations as sodium and
potassium levels are tightly regulated at the level fo the kidney.
Perhaps with more power to test (ie. different experimental design) the
detection of different net absorption rates might be more likely.

Chloride was unaffected by monensin supplementation, and the data
indicates that chloride is taken up by the intestine (negative net
absorption rates (Table 16).

I was also unable to detect differences in either blood L-lactate,
blood glucose and total blood lipid concentrations or their P-A
differences with monesin. L-lactate concentrations in portal blood
(Table 11) decreased from .961 mM to .917 mM and net absorption values
declined from 15.60 mM/hr to 6.68 mM/hr. This is in agreement with
data obtained by Harmon et a1. (1986) and is likely to be a reflection
of a decreased ruminal production of lactate, a well documented
monensin effect on the rumen (Bergen and Bates, 1984). However, the
data is contrary to other data reported by Huntington and Harmon (1986)

and Harmon and Avery (1986). Neither of these studies showed any

100
significant differences in L-lactate absorption with monensin
supplementation. Table 19 compares published data to the data of this
study.

Glucose concentrations were also not different although there was
a tendency for monensin supplementation to increase portal glucose
concentrations. This finding is also in agreement with Huntington and
Harmon (1986), Harmon and Avery (1986) and Harmon et al. (1986). The
data of the other studies in Table 19 do indicate a tendency fer
monensin to increase glucose uptake which might be explained by an
increase in Na+/K+-ATPase activity with monensin supplementation.
However, none of these previous studies were able to detect a
significant increase in glucose uptake.

Blood alpha-amino nitrogen (AAN) and blood urea nitrogen (BUN)
concentrations were not significantly afffected by monensin
supplementation (Table 12). .AAN values were higher for monensin
supplemented cattle than control cattle. BUN was lower fer monensin
supplemented than control steers. These data are also in agreement
with previous studies (Table 12). Net absorption rates for AAN
increased from 187.18 mM/hr to 226.47 mM/hr, while BUN net absorption
rates decreased.

Portal blood flow rates were not signifantly altered with monensin
supplementation. The portal blood flow rates observed in this study
are similar to rates obtained by others (Huntington, 1983; Huntington
et a1, 1981) for steers of this size on a high energy diet. They are
also in agreement with all studies except the Huntington and Harmon

(1986) study. This may be a result of the way in which the studies

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l02
were conducted. Huntington and Harmon (1986) utilized a switchback
design to measure the effect of monensin removal. They therefore
measured an acute situation, rather than a chronic situation as
measured by other studies.

From these data and those reported in Table l9, it is obvious that
there cannot be a clear picture of the effects of monensin
supplementation in beef cattle. There seems to be some evidence of an
effect in the rumen (magnesium transport) and an effect on some
minerals but no clear effect on intestinal absorption processes that
might be altered by known mechanisms of monensin action (i.e. sodium
dependent processes). It may be that monensin is having an effect that
is unmeasurable by our present techniques or that alterations do occur
but the intestine buffers those alterations in a homeostatic
mechanisn.

These data when coupled with the significant increase in
Na+/K+-ATPase activity in mucosal biopsies from monensin
supplemented steers indicate that monensin does have an effect at the
cellular level. Our inability to detect significant differences in
sodium-dependent processes do not preclude them from occurring.
Further studies of monensin absorption in the intestine should couple
in vitro examinations of the effect of monensin on the enterocyte with
infusion studies in the animal.

Intracellular alterations of monensin may provide more insight
into metabolism of the cell. It may be that ions are altered in their
sequestration patterns in the cell. Metabolic pathways and processes

may also be altered. Another important consideration is the unstirred

103

water layer. To what concentration is the cell exposed to This is an
important consideration in absorption studies and one that needs to be
addressed.

Further examinations of net absorption should involve either
monensin instions into the small intestine to examine intestinal
effects or, the rumen venous drainage should be cannulated along with

the portal vein. This would help partition out the effect of monensin

in the rumen from that in the snall intestine.

APPENDIX

100)

consumption (inhibition/
3 M ouabain x

104
2

inhibition at 10

Inhibition of mucosal 0

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caucus: nozomsaxmflsoz

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Literature Cited

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