methods

in nutrition

Polyethylene glycol as a quantitative fecal marker in human nutrition experiments13 Lindsay

H. Allen,

Ph.D.,

William

Data

ABSTRACT was for

fed to 5 1 subjects of

of the subjects. marker

for

faster

42

Fecal

days

showed

marked

However, further for

average

fecal

is needed

the correction

output problem

of test nutrients

from

ceased.

intestinal recovery

before

balance

body.

The

of measuring intakes accurately has been greatly simplified by the use of liquid formula diets. However, it is still difficult to obtain precise fecal output data for the following reasons. 1) for an unknown period after feeding an experimental diet, the feces

will contain nutrients ingested before the test diet was fed; 2) some individuals “pool” feces within their intestines for long periods of time. Without the inclusion of a fecal marker, this might be interpreted as reflecting a high rate of absorption of the test nutrients; 3) after termination of feeding an experimental diet, there exists an unknown before the unabsorbed portion

excreted; and underestimated tion.

4) fecal output due to losses

period of time of that diet is

is frequently during

This

only

collec-

zinc,

fed

Inclusion diet and

of only

93% Am.

data.

PEG

J. C/in.

the

in

or

Cr2O:,

consumed

can

be used

32: 427-440,

was

excreted

markers of

fecal In one

sporadically

eliminated,

markers Nutr.

sodium.

PEG

the

in 50%

was a valid

excreted

amount

when

to occur

for

fecal

completely

changes

of these

PEG

were

quantitative

been

of the

of either

excreted

less valid

(PEG)

fed continuously

7 days

consumed. was

of Cr2O,

of

had

than

simultaneously,

amounts

glycol

was

completely

more

but

were

small

PEG

been

took

93% of that

and

contents,

the recovery

rely on accuinput and the

the

to have

M.D.

polyethylene

experiments

assumed

preexpenmental

of nutrient

Nutritional balance studies rate measurments of both the

had

in which

In three

(Cr2O:i)

subjects,

Margen,5

experiments

iron,

oxide

Sheldon

constant.

averaged

In most

feeding of

was

became

chromic

when

pooling

an work

time

five

marker.

magnesium,

Cr203.

Cr2O3

of the

diet of PEG

and

than

after

identification

from

fecal

solids

recovery

PEG

average

dry

calcium,

where

on

for

to fecal

nitrogen,

experiment

summarized

Preexperimental

PEG

fecal

are

M. S., and

as a quantitative

106 days.

35 to

ratio

L. Raynolds,4

fecal with

permitted subjects

who

dry

solids.

suggests

that

confidence

1979.

nutrition research unit. The laboratory analysis of fecal markers is expensive and timeconsuming, so we will attempt to assess the value of using them in several types of experiments. Most of our experience has been with the marker polyethylene glycol (PEG), although chromic oxide (Cr203) was used in one experiment. The criteria for validity of a material as a fecal marker have been described by others (1). Both PEG (2) and Cr203 (3) have been stated to meet these criteria.

Methods The ducted I

versity 2

data

were

derived

over

3 years

in

from our

human

five

experiments nutrition

conunit.

All

From the Department of Nutritional Science, Uniof California, Berkeley, California 94720. Scientific Contribution no. 702, Storrs Agricultural

The purpose of this communication is to summarize our fmdings to date on the usefulness of including quantitative fecal markers in the diets of subjects in our human

Experiment Station, University of Connecticut, Storrs, Conn. 06268. J Address reprint requests to: Dr. Lindsay H. Allen, Associate Professor, Department of Nutritional Sciences, University of Connecticut, Storrs, Conn. 06268. 4 Assistant Specialist in Nutrition. Professor of Human Nutrition.

The American

1979, pp. 427-440.

Journal

ofClinical

Nutrition

32: FEBRUARY

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Printed

in U.S.A.

427

ALLEN

428 subjects containing source, solution

received known amounts ofliquid formula diets, egg albumin or isolated soy as the protein maltodextrins, cornstarch and corn oil. PEG (polyethylene glycol, MW 3000 to 3700, Matheson, Coleman and Bell, Norwood, Ohio), was added to each subject’s food container at each meal and served at 0830, 1230, 1630, and 2030 hr. Adequate minerals and vitamins were supplied. Deionized water was available ad libitum and the amounts consumed recorded. The body weights of subjects were maintained to within ±2 kg of their initial weight by means of a calorie supplement when necessary. This supplemental formula contamed maltodextrins, cornstarch, sucrose, corn oil, and water. Dietary nutrients were analyzed in composite samples obtained from complete daily intakes of all food items. Urine and fecal samples were collected daily (experiments IV and V), pooled for 3 days (experiments II and III), or pooled for 3 and 4 days alternately (experiment I). These samples were analyzed for the same nutrients as the diets. The methods of sample preparation and nutrient analysis have been described elsewhere (4). Completeness of urine collection was continuously monitored by measuring urinary creatinine excretion.

Analyses PEG was analyzed in diets and feces by an adaptation of the method of Malawer and Powell (5). In experiment II, some subjects had poor absorption of protein from a high protein diet (36 g nitrogen per day) during an initial period when egg albumin was the protein source. The increased fecal excretion of protein interfered with the analysis for PEG. Smith (6) reported the presence of substances, thought to be protein, in the calf alimentary tract which interfered with the determination of PEG. Based on his observation that mercuric chloride eliminated interference by protein, the PEG assay was modifled as follows; 1.0 ml water (blank), or 1.0 ml PEG standard (0.5 to 14.0 mg/ml), or 1.00 g of fecal homogenate was added to 25 ml Erlenmeyer flasks. To these were added 2.0 ml of albumin solution containing 150 mg/I, 2.0 ml of 15% BaC12, 6.0 ml ofO.3 N Ba(OH)2, 1.0 ml of 30% ZnSO4 .7H2O, and 1.0 ml of 5% HgCI2. After

TABLE Summary

ET

AL.

vigorous shaking, and standing for 10 mm, samples were filtered through Whatman no. 2 filter paper. To 1.0 ml aliquots of the filtrates were added 3.0 ml of solution containing 12 mg/mI gum arabic, and 4.0 ml 30% trichioroacetic acid. After mixing, and standing for 60 mm, OD was measured in a spectrophotometer at 650 mi with a slit width of 0.04 mm. These concentrations of reagents gave PEG recoveries of 98.3%, (SD = 1.26%) when known amounts of PEG were added to feces. One fecal sample containing PEG was analyzed 20 times over several months; the coefficient of variation was 1.7%. Chromic oxide (Fisher Scientific Company, PiUsburgh, Pa.), was measured after the method of Bolin et al. (7). Fecal aliquots were digested with a solution containing seven parts concentrated nitric acid, 1 .75 parts 70% perchloric acid and 0.25 parts concentrated sulfuric acid. The resulting yellow solution (dichromate) was read in a spectrophotometer at 440 m.t. Experiments A summary of the five experiments in which PEG was used is provided in Table 1. In the first three experiments, PEG was fed “continuously” for 35 to 105 days. In experiment IV, PEG was fed on only 1 day, and in experiment V, for 6 days. In those experiments where PEG was fed continuously (I to III), it was used to determine the point at which preexpenmental diets had been eliminated. As will be discussed later, this point was assumed to have been reached when the concentration of PEG in fecal dry solids became constant. In experiments I and II, the nutrients excreted in feces during the study were corrected for PEG which had been fed, but not excreted, i.e., total fecal excretion of nutrient X (total PEG fed + total PEG excreted.) This correction enabled us to account for diet still retained in the intestine at the end of the experiment. In experiment III, this correction was made for each 3-day fecal pool in order to correct for day-to-day variation in fecal flow, using the formula fecal zinc (mg/day) x (diet PEG (mg/day) + fecal PEG (mg/day)). In experiment IV, PEG was fed on I day only, together with the test nutrients, in order to follow the time and completeness of their excretion. In experi-

1 of experiments

Experiment

Subjects/sex

Topic

Days when PEG administered

ert

Purpose

of feeding

PEG

days

I

OCA

II III

Protein OCA/Zn

IV

OCA/Cu,

V

Zn

deficiency

Fe,

Zn

6/F

I 17

1 1-1 17

To

6/M 10/F

105 35

1-105 1-35

To

23/F

9-12

4

6/M

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15-18

1-6

detect perimental retention ment.

feces derived from preexdiets, and to correct for of diet at end of experi-

detect feces derived from preexperimental diet, and to correct for 3-day variations in fecal flow. To follow excretion of experimental diet. To detect feces derived from preexperimental diet.

HUMAN ment

V,

PEG

was

fed only

in the

preexperimental

NUTRITION diet,

and the time of its last appearance in feces taken as the time when preexperimental diet had been eliminated. Experiment I was a study of the effects of oral contraceptive agents (OCA) on the nutrient balance and metabolism of six young women (8). The study lasted for 1 17 days, with each subject receiving OCA for part of the experimental period. From day 1 1 to day 117, 1800 mg/day PEG was administered as a solution added to the diet. In addition, analysis of the vitamin pills showed that these supplied an additional 100 mg PEG per day, mostly from the choline supplement. The fact that PEG is used as an inert filler in many vitamin and mineral supplements has been reported by other investigators. During days I I to 25 of this experiment another fecal marker, Cr2O:t, was also used. Three hundred milligrams ofCrsO:i per day was mixed with a small amount of sugar and fed with the formula diet. Diets, urine and feces were analyzed for nitrogen, calcium, magnesium, sodium, potassium, iron, copper, and zinc. Fecal dry solids, and PEG and Cr2O3 in feces and diet, were also determined. Experiment II was an investigation of the effects of high protein diets on the metabolism and nutrient balances of six young men, 23 to 30 years old (4). During the 105 day study, each subject consumed a 12 g of nitrogen (12 g N) diet for one half of the experiment, and a 36 g of nitrogen (36 g N) diet for the other half of the period. Each subject ingested 1600 mg of PEG per day, added to each meal as a solution. An additional 109 mg of PEG per day was ingested in the vitamin supplements, so that the total intake of each subject was 1709 mg of PEG per day. Diets, urine, feces, and other excreta were analyzed for nitrogen, calcium, magnesium, sodium, and potassium. Dry solids were measured in feces, and PEG in both diets and feces. Experiment III involved the feeding of PEG to 10 young women for 35 days, in an attempt to measure the effect of OCA on endogenous zinc excretion (9). A zincdeficient diet (0.2 mg zinc per day) was fed throughout the experiment. When the concentration of PEG in dry solids became constant, it was assumed that all diet ingested before the experiment had been excreted, and that all fecal zinc was of endogenous origin. As previously described, the ratio of fecal PEG to fed PEG was calculated for each 3-day fecal pool in order to correct for day-to-day variation in fecal flow. Experiment IV was designed to measure the absorption of stable isotopes of copper, iron and zinc in women who were or were not taking OCA ( 10). A formula diet was fed throughout the 12-day study. The trace elements Fe, Cu, and Zn were added by solution to each meal so that Il mg ofFe. 3 mg ofC#{252}, and 11 mg ofZn were provided daily. On day 4 of the study, stable isotopes of these elements, Fe, Cu, and Zn7#{176}, were fed in place of the naturally occurring isotopes at the same level. Apparent absorption was calculated from dietary and fecal analyses by neutron activation. In this study PEG was used to monitor the time at which the transit of the isotopes through the intestine was complete. On the day of administration of the stable isotopes (day 4), 2000 mg of PEG was divided equally among all four meals. PEG was measured in subsequent fecal samples. When PEG recovery was complete, transit of the isotopes was assumed to be complete. In a few cases where PEG recov-

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429

EXPERIMENTS cry

was

incomplete

at the end

of the

12-day

period,

the

percentage recovery of PEG was used to estimate the amount of unabsorbed isotope retained in the intestine. Experiment V was an investigation of the effects of a zinc-deficient diet on biochemical parameters and endogenous zinc excretion in six young men ( I 1 ). A standardization diet containing 15 mg of Zn per day and 2000 mg of PEG per day was fed to all subjects during the first six days of the experiment. At this point the diets were changed so that they contained no zinc or PEG. When fecal PEG concentrations dropped to zero, it was assumed that all fecal zinc was endogenous in origin.

Results As expected, the day-to-day variation in fecal flow was considerable. Wet solids ranged from 0 to 1 17 g/day, and dry solids from 0 to 39 g/day. Without the inclusion of fecal markers, this sort of variation makes it impossible to obtain accurate balance data over short periods oftime. Figure 1 shows the excretion of dry solids and PEG by one subject (3202) in experiment I. This subject cxcreted twice as much fecal material in the last half of the experiment compared to the first, in spite of a constant intake of dry matter. The ratio of PEG to dry solids became constant in each subject, after an initial equilibration period when PEG mixed with intestinal contents ingested before PEG was fed, i.e., before the experiment started. Figure 2 shows this equilibration of PEG with fecal dry solids for subject 3201, and the concentration of each nutrient relative to PEG at equilibrium. These concentrations are different for each subject, but are relatively constant in each individual. By plotting the data for each subject as shown in Figure 2, the point at which equilibrium is reached can be judged by eye. Using this approach, it was decided that subject 3204 took 4 to 7 days; 3201, 3202, 3205, and 3206 took 8 to 10 days; and 3203 took 15 to 17 days to reach equilibrium. Using a statistical approach, this point was also determined by fitting a broken-line regression equation to the data by the method of least squares (12). By this method, it was calculated that the time taken to reach equilibrium was the same as judged by eye for subjects 3201, 3204, 3205, and 3206, but that it was 14 days longer for 3203, and 32 days longer for 3202. However, the plateau concentration of PEG in dry solids calculated by regression was only very slightly higher in the

ALLEN

430

ET

AL.

0.

0

DRY SOLIDS

PEG

g/day)

(g/day)

60-

.6

40.

.4

2

20

1’

I;

I 0

...0 I 20

80

40 DAYS

FIG. 1. Excretion 11 today 117.

day

of fecal

dry

solids

and

PEG

for subject

in experiment

3202

I. PEG

was

fed in every

meal

from

in every

meal

200

150

U) 0 -I

0 U) ).

100 nutrl.nt/9

nag

0 0

Mg:1

Ui

a. ci

50

.quilSbrium

341 :239

Ca

0

at

PEG

N :

08

Fa

:11

Zn

:9

A-

0

40

2’O

6’O

80

tOO

120

DAYS

from

FIG. 2. Equilibration day 1 1 to day I 17.

latter

two subjects

estimation

Some

of the

PEG

of PEG

with

than that equilibrium

appeared

in

fecal

dry

solids

calculated after point by eye.

the

first

3-day

pooled fecal sample ofeach subject after PEG feeding started. However, since it took considerably longer than this for all the previous

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for subject

3201

diet of

in experiment

to be excreted the

marker have diffused intestine soon

danger fecal

and along after

I. PEG

(from

was

4 to 17 days),

some

experimental diet must the entire length of the it was fed. There is a

that this also happens such as the

markers

fed

with qualitative commonly used

HUMAN

NUTRITION

carmine red and brilliant blue. Our data show that the mere presence of a marker in feces does not mean that all feces formed before

the experiment have been eliminated from the body. Figure 3 shows the cumulative recovery of PEG for all subjects in experiment I. Recovcry approached 100% of that fed in varying amounts of time, depending on the subject. Only one subject (3203) excreted 100% of the

PEG Most

she was given over the 106-day period. subjects began to approach 100% recovcry of PEG within a week of its initial consumption. ject 3202

However, the fecal output of subwas so low in the first half of the

experiment

that

it took

60 days

to approach

intake.

Without

PEG, during the excretion

the first 60-day

for excretion

the inclusion

metabolic

of

period

of all nutrients in the feces of this subject would have been underestimated by 50%, and in the other subjects by from 5 to 30%.

In addition uously

from

to PEG, days

Cr203

was fed contin-

1 1 to 25 of experiment

I.

431

EXPER1MENTS

However, fecal excretion sured until day 46 in one experiment at this point), subjects, and until day ject. The cumulative throughout the experiment 4. One hundred percent achieved in one subject, excreted only 88 to 95%

In

all

subjects

there

of Cr203 was measubject (who left the until day 67 in four 105 in the sixth subrecovery of Cr203 is shown in Figure recovery was only while the other four of the total intake.

was

still

some

small

excretion of Cr203 throughout the entire penod during which Cr203 excretion was followed. Even 45 days after Cr203 feeding ceased, subject 3202 was excreting considerable quantities of Cr203. There were periods, however, from 3 to I 5 days in duration, during which the feces contained no Cr203. This fact emphasizes the difficulty of determining when all Cr203 in the body has been excreted. PEG and Cr2O3 were fed simultaneously from days 1 1 to 25. This gave us the opportunity of comparing the excretion of these two markers. These data are plotted in Figure 5, which shows the percentage recoveries of

>. Ui

> 0 0 Ui

C, Ui

a.

0

3201

.

3202

a

3203

V

FIG. 3. Percent recovery of cumulative every meal from day I I to day I 17.

dietary

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PEG

in feces

of all subjects

3204

a

3205

0

3206

in experiment

I. PEG

was

fed

in

ALLEN

432

ET

AL.

100

0

3201

.

3202

a

3203

V 3204 0 3206 0

3206

A-

20

0

40

&0

i6o

8’O

io

DAYS FIG. every

4. meal

Percent from

day

recovery of cumulative I 1 to day 25.

dietary

CrsO:i

cumulative dietary PEG and Cr2O3 during this time. In three of the six subjects (3204, 3205, and 3206) the recovery of Cr2O3 was consistently less than that of PEG. In one subject (3201) the recovery of Cr203 was consistently higher, in another (3202) the recovery of Cr203 was lower until the last fecal sample. When the PEG and Cr203 data from all subjects in this experiment were combined, the total excretion of PEG was significantly

greater (using

than

that the

of Cr203

paired

during

Student’s

days

I 1 to

P < 0.01). Because it is water soluble, PEG is thought to resemble most ions more closely than does insoluble Cr203. In order to test for those nutrients for which PEG is a valid fecal marker, the ratios of several nutrients to PEG were determined in each fecal sample of six subjects in experiment I. Significant correlations (P < 0.001) were found between the 25

I

test,

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in feces

of all subjects

in experiment

I. Cr203

was fed with

amount of nitrogen, calcium, magnesium, iron, zinc, and the amount of PEG in fecal samples (Table 2). However, the correlation of PEG with sodium was less significant in one subject (P < 0.01), and not significant in two subjects. Experiment

II

PEG was also fed continuously in this cxperiment. Five of these subjects took from 1 to 3 up to 10 to 12 days to completely excrete diet ingested before the experiment, i.e., for the PEG concentration in feces to become constant. However, the fecal output of subject 3205 was small and irregular, so this subject took 39 days to reach equilibrium. Final recoveries ranged from 91 to 104%, average 94 ± 2.3%. Overall nutrient balances were corrected for PEG not yet excreted in the same way as in experiment I. In this experiment, the average daily excre-

HUMAN

NUTRITION

433

EXPERIMENTS

tion of fecal dry solids was 19 to 45% (average 32%) higher when subjects were changed from the 12 g N to the 36 g N diet, presumably due to the inclusion of the additional 24 g N as isolated soy protein. If fecal excretion of each nutrient had been expressed in terms of its concentration per gram dry solids, as is frequently the case, it would have appeared as though the apparent absorption of each nutrient had been increased by the consumption of the high protein diet, i.e., the nutrient concentration per gram dry solids was decreased. However, when the data were ex-

pressed

PEG

as nutrient

in feces,

concentration

there

was little

ratio as a result of a change shows the fecal excretion pressed in relation to both and PEG. Experiment

per

change

gram

in this

in diet. Figure 6 of calcium exfecal dry solids

III

It was assumed in this experiment that when PEG excretion per gram of dry solids became relatively constant, prestudy zinc was cleared from the intestinal tract, and fecal zinc was therefore ofendogenous origin. This

100-

3202

3201

3 203

>.

w >

0 0 w

I-.

0.

t1

I

100-

z

w 0

3206 3204

‘U

a_

0-%

,

0

#{149}

10

15

1

#{149}

20

,It1I0

25

1

1

25

110 hhu115

DAYS FIG. 5. Comparison all subjects in experiment

TABLE Correlations

a

percent

recovery

I. Both

PEG

and

of cumulative dietary PEG (#{149} Cr203 were fed with every meal from

#{149}) and day

CrsO:t (D-{J) I I to day 25.

in feces

2 of fecal

nutrients

fecal

except

those

PEG 3202

23 0.929’ 0.957 0.954 0.879 0.889 0.749 0.938 0.863

of samples Wet solids Dry solids N Ca Mg Na Fe Zn All correlations

with 3201

Subject

No.

of

27 0.945 0.98 1 0.957 0.987 0.893 0.645 0.988 0.983 ma rked

otherwise

were

Downloaded from https://academic.oup.com/ajcn/article-abstract/32/2/427/4666291 by University of Glasgow user on 02 August 2018

3203

3204

29 0.676 0.873 0.786 0.841 0.764 0.3296 0.857 0.790

29 0.806 0.937 0.661 0.921 0.845 0.3406 0.773 0.906

significant,

P < 0.001

3205

24 0.845 0.93 1 0.850 0.881 0.921 0.534’ 0.929 0.910 .

“

Not

significant.

3206

9 0.947 0.976 0.956 0.981 0.989 0.931 0.982 0.982 P < 0.01.

of

434

ALLEN

ET

AL.

0

E an

&

..p... 9

(ow) 93d/

(bw)

0

1flI31V3.----.

0

a

w

-5

an

0

E an

a

E C “I

‘O

0

0

‘O 1

0

t

E an

& 0

S

I.-

0

E an ci

en

E

I

0

C U

E

.

& U C

U U

0

0

:---

.0

C 0

a

U U

?

U

?

E U

(6)SGflOS

AJ0

1V03d/ibW)

vgnIolv3-.-

U

U

LI,

0.

9

Downloaded from https://academic.oup.com/ajcn/article-abstract/32/2/427/4666291 by University of Glasgow user on 02 August 2018

HUMAN

NUTRITION

occurred by the third pooled fecal collection of all subjects, on days 8 to 10. Fecal zinc from day 8 onward was then corrected for the recovery of PEG in each 3-day fecal sample, using the formula previously described. This

enabled the construction the endogenous fecal

ofsmooth

curves

be expressed

zinc

solids

during

the absence in dry

excretion deficiency

(Fig.

7). As illustrated

in

experiment II, the danger of this approach is our inability to correct for a change in fecal dry solids that might result from a change in treatment. In experiment III, fecal dry solids were not significantly less in the presence of OCA, although there was a trend in this direction. With more subjects or a longer experimental period, the data might have shown that endogenous zinc excretion relative to PEG was lower in the subjects on OCA, while endogenous zinc concentration per gram dry solids was not changed. Final recoveries of PEG ranged from 84 to 105%, averaging 94% (SD = 7.1). Since this

.40

.30

a

-0CA

.

+OCA

435

50 n.9

Cl) 0 UI

n.6

for

of zinc (mgi the zinc (Fig. 7). In of PEG, the data would have to in terms of the concentration of

day)

EXPERIMENTS

Cl) 25U.

0 I,-

z

nx2

UI 0

n-I

UI An 1-3

8-40

11.43

14.47

39.42

DAYS FIG. 8. Time taken for subjects in experiments III to complete excretion of preexperimental diet. were combined from 20 subjects.

was was

I to Data

not a balance made for the

experiment, no correction overall recovery of PEG. In experiments I to III, PEG was fed for a sufficient length of time for its concentration in fecal dry solids to become constant. Figure 8 is a summary of data from 20 subjects in these three experiments, showing the time at which this occurred, i.e., the time at which the preexperimental diet was completely eliminated. Experiment

0

4-7

I V

.20

PEG was fed only on day 4 of this study, at the same time as the test meals of stable isotopes. Out of 23 subjects, 14 (61%) began

a .10

.

Q ;

0

V

Q

to excrete collected

U I

.60

.50

a

-

.

+ OCA

OCA

.

a .40

:

D S

N

subjects subjects purpose

a

a

.3C

#{149}

D S

‘o

2’O

#{149}

‘o

DAYS

FIG. 7. all subjects grams ofZn Zn corrected fecal pool.

Fecal zinc excretion; average of values from in experiment III, expressed both as milliper gram ofdry solids, and as milligrams of for percentage recovery of PEG in each

Downloaded from https://academic.oup.com/ajcn/article-abstract/32/2/427/4666291 by University of Glasgow user on 02 August 2018

PEG

after

in the first

PEG

was

3-day fecal pool given, while seven

(30%) took from 4 to 6 days, and two (9%) took from 7 to 9 days. The of feeding PEG was to determine the time when the fecal excretion of PEG, and therefore the stable isotopes, had ended. This took from I to 3 days in one subject out of 20 (5%), 5 days in two subjects (10%), 7 to 9 days in eight subjects (40%), more than 9 days in eight subjects (40%), and more than 12 days in one subject (5%). For most of the subjects, fecal PEG analyses were done for 9 days after PEG was fed. During these 9 days, PEG recovery ranged from 48 to 90% (average 83 ± 10.6%). The two subjects who took from 7 to 9 days to start excreting PEG only excreted 48 to 55% of the PEG at the end of 9 days.

436

Experiment

ALLEN

V

PEG was fed during the first 6 days of this experiment, at which time both PEG and zinc were excluded from the diets. PEG appeared in the first 3-day pooled fecal sample from 5 of the 6 subjects, and in the second 3-day sample (days 4 to 6) of one subject. One subject took from 13 to 15 days until fecal samples no longer contained PEG, and the other five subjects took from 16 to 18 days. Final recoveries of PEG were from 79 to 104%, average 93 ± 8.6%. In some subjects we analyzed fecal samples after the PEG content first fell to zero, but in none of these subsequent samples could we detect PEG. Discussion In nutrition balance experiments, two criteria have traditionally been required before a subject can be said to be in “steady-state” with respect to the experimental diet; 1) any diet ingested prior to the experiment must have been excreted, and 2) the rate of excretion of a fecal marker must approach its rate of intake. Balance data from subjects who do not fulfill these criteria have been regarded as unacceptable (3). With regard to the first criterion, various methods have been used to deduce when the preexperimental diet has been excreted. 5everal investigators have simply assigned a certam number of days as an “equilibration period” for this to occur, e.g., 5 or 6 days, (2). Some administered a qualitative colored fecal marker at the same time as the quantitative marker (13-16), but as we report in this paper, the mere appearance of a marker in feces does not mean that it is equilibrated with the feces. A similar concentration of the marker in feces during two consecutive collection periods has also been used as the criterion (15-17).

We have defined the time at which the concentration of fecal marker in dry feces becomes constant as the time when the previous diet has been eliminated. For all practical purposes, we have found that this point can be estimated by eye after plotting the data, although broken-line regression analysis could be used for this purpose. Davignon et al. (3) used the concentration per gram of wet feces, but the concentration of water in feces varies markedly and is not

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consistent from one collection period to the next. It took from 8 to 10 days for 80% of the subjects in experiments I to III to reach a constant concentration of PEG in dry fecal solids. This means that most subjects in our balance experiments were excreting preexperimental diet for at least a week after the experiment started. In studies lasting for less than a week, half of the subjects would have their feces contaminated by diet ingested prior to the experiment. While it is conceivable that liquid formula diets might slow down fecal transit time (18), the intestinal contents from food ingested before the experimental diet were actually derived from “normal” foods. Similar results were found when PEG was fed for only a short period of time (experiments IV and V) and the length of time for PEG to disappear from feces was recorded. In experiment IV, 45% of the 20 subjects took longer than 9 days, and 5% longer than 12 days before fecal PEG was zero, while in experiment V, five out of six subjects took 16 to 18 days for this to occur. The second criterion which has been used to defme the attainment of steady state is that the cumulative recovery of PEG should be close to 100% of the amount fed. Davignon et al. (3) arbitrarily defmed a cumulative recovery of 90% as being acceptable. Certainly subjects who have long fecal transit times, and, therefore, low recoveries of marker over the short-term, might be regarded as “nonideal.” However, by inclusion of PEG we have detected at least one subject in each of these five experiments who was very slow to excrete the experimental diet, all of whom came into equilibruim by our definition (a constant ratio of PEG to fecal dry solids). There does not appear to be any valid justification for excluding balance data from those subjects with cumulative recoveries of PEG less than this arbitrary 90% if equilibrium has been established. Once the ratio of each nutrient to PEG at equilibrium has been established, several uses can be made of this information. The ratio of each nutrient to PEG in the diet compared to its ratio to PEG in feces can be used to estimate the apparent absorption ofthat nutrient. For example, subject 3201 consumed 303 mg of calcium per gram of PEG in the diet, and at equilibrium excreted 239 mg ofcalcium per gram of PEG. The difference in these ratios reflects appar-

HUMAN

NUTRITION

ent absorption of calcium, which for this subject was calculated to be 21 I %. After equilibrium, any change in the ratio in a single fecal sample usually signified an analytical error that could be corrected by repeating the analysis. If necessary, the ratio can be used to calculate how much of each nutrient excreted in feces before equilibrium is from the experimental diet, and how much is from the preexperimental diet. For examplc, subject 3602 (experiment II) excreted 263 mg of N per gram of PEG at equilibrium (days 21 to 1 17), but 430 mg of N per gram of PEG during days 15 to 18. We can therefore assume that approximately 263i430 = 61% of fecal nitrogen excreted during days 15 to 18 was from diet ingested before the time when PEG feeding commenced, and that 39% of the fecal nitrogen excreted during this time came from our experimental diet. Similar calculations could be made at the end of an experiment of this type where the excretion of PEG in feces has become constant; an estimate can be made of how much of each nutrient in the fecal sample is from the experimental diet, and how much is from foods consumed after the consumption of PEG and the experimental diet has ceased. This could not be calculated in experiments I to III since fecal samples were not collected after PEG feeding ended. One major purpose of feeding PEG is to correct balance data for incomplete recovery of PEG, with the assumption that this represents retention of feces within the intestine after fecal collections have been discontinued. However, in our studies, overall recoveries of less than 90% occurred frequently. In experiments I to III where PEG was fed for relatively long periods of time, and feces were not collected after PEG feeding has ceased, the overall cumulative recovery of PEG was 92.5 ± 7.7%. The 7.5% not recovered amounts to a large quantity of PEG that would be unlikely to be retained in the intestine at the end of the experiments. In experiment V, after PEG had been fed for a short period of time, feces were analyzed until the PEG concentration fell to zero. Cumulative recovery in this experiment was 93. 1 ± 8.6%, which was similar to that found in the previous experiments. Our low cumulative recoveries of PEG could be due to several factors; 1) overesti.

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mating intake, which would be unlikely to occur to this extent with liquid formula diets and other precautions taken to ensure complete intakes, 2) loss of feces on toilet paper, although this has been estimated to be a relatively small loss (15), 3) errors in our analytical technique, despite our ability to recover 98. 1 ± I .26% of known amounts of PEG added to feces, and 100% of the PEG fed to some subjects, 4) retention of the PEG within the intestine in experiments I to III since feces were not collected after PEG feeding stopped, 5) absorption of small amounts of PEG by the intestine, or 6) destruction of small amounts of PEG by bacterial or intestinal enzymes. Only a few investigators have measured the cumulative recovery of PEG in human subjects. PEG 4000 (MW 3000 to 3700) was used in all these experiments. The Food and Drug Administration has approved PEG for inclusion in human diets if its molecular weight is greater than 200 (19). Wilkinson (2) fed 1500 mg of PEG 4000 to six subjects for 14 days then collected additional feces until the PEG was completely excreted. The mean recovery was 98.6%, range 95.5 to 101.5%. In two ofthese six subjects only 95% ofthe PEG fed could be recovered. In the second part of Wilkinson’s study, 64 patients were fed 1500 mg PEG daily for an “equilibration” period of 7 days, followed by a balance period of 7 days. The recovery of PEG, expressed as a percentage of the dose administered in the last 7 days, ranged from approximately 27 to 180%, average 99.3% (SD 23.6%). The conclusion was drawn that since PEG recovery in the last 7 days was normally distributed and had a mean of99.3% ofthe administered dose, then PEG was completely recoverable in the feces and was being estimated accurately. However, there was no evidence that a prebalance period of 7 days was sufficiently long to achieve equilibration, since the ratio of PEG to dry solids was not measured. Indeed, the fact that many patients had not equilibrated during this period is demonstrated by their excreting far more than 100% of the intake during the next 7-day balance period. The 7-day equilibration period appears to be arbitrary; if it had been shorter, the mean recovery for all subjects in the balance period might have been less than the observed 99%, and if it had been longer,

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mean recovery might have been more than 99%. In either case, the mean recovery of PEG calculated in this manner reflects the mean rate of fecal flow and excretion of PEG, rather than the ability to accurately recover 100% of what was fed, or proof that PEG had not been absorbed from or destroyed in the intestinal tract. Shields et al. (20) achieved 100. 1% (SD ± 2. 1) recovery of PEG 4000 administered during a 90 to 180 mm period to 1 1 patients by colonic infusions. However, the collection of rectal effluents was complete within 4 hr of starting the experiment. The possibilities of PEG being degraded in the intestine over a longer period of time, or of it being absorbed higher in the intestine, are not excluded by this experiment. Beeken (2 1) fed from 2 to 4 g of PEG 4000/day for up to 8 days, and analyzed fecal PEG from days 4 to 8. During these 4 days, recovery of PEG was only 70% of that fed during the same period. This low recovery must be partly due to continued excretion of the preexperimental diet, and to fecal retention of the PEG fed. In two normal subjects, Soergel and Hogan (22) recovered 97.0 and 96.3% of 2 and 3 g of PEG given orally. In six fasting ileostomy patients given 15 g of PEG 4000 orally, they recovered 96.9 ± 2.8 (mean ± SD, range 92.9 to 100.4%) ofthe dose. The mean transit time ofPEG was 1.5 hr. We must conclude, therefore, that PEG recoveries of less than 100% are not uncommon. Further work is needed to determine the reason for this before we can be confident of correcting nutritional balance data for nonrecovery of PEG. In addition, there is still a possibility of intestinal or bacterial degradation of PEG in some subjects, and the absorption of PEG or its breakdown products. Our experiments suggest that Cr2O3 was excreted slightly more slowly than PEG. Findlay et al. (23) found that these two markers move similarly in subjects with normal bowel habits, and concluded that “streaming” of intestinal content does not usually occur, i.e., there is no difference in the flow rate between the liquid phase (for which PEG is a marker) and the solid phase

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(for which Cr203 is a marker) of the feces. Wilkinson (2) compared the excretion of PEG with that ofCr2O3 of 1 1 patients. During the first 2 weeks that the markers were fed simultaneously to these patients, PEG was excreted faster than Cr203 but during the 3rd week the markers were excreted at a similar rate. However, within 5 days after discontinuing marker administration all PEG had been excreted, while at 7 days Cr2O3 was still being excreted. Small amounts of Cr203 were still being excreted for 42 days after feeding ceased in four of our six subjects, and for 80 days in one of them. The absence of Cr2O3 in some of the intervening fecal samples during this time suggests that the relatively dense Cr2O3 sediments in the intestine and is therefore excreted irregularly. In contrast, once PEG in feces fell to zero it was not observed in subsequent samples. Whitby and Lang (17) achieved 93% recovery ofCr2O3 within 3 days of its administration, and assumed that since Irwin and Crampton (24) found that the passage of Cr2O3 ended within 48 hr of the last ingestion, this was the final recovery to be expected. Rose (13) obtained an overall recovery of 93% (range 80 to 103%) in 21 studies, while Davignon et al. (3) had a total recovery of 98.7 ± 5.8% from six subjects. While we followed Cr203 excretion after feeding ceased for a much longer period of time than other investigators, we achieved from 89 to 95% final recoveries in four out of six subjects, and close to 100% in only two of these subjects. Since it seems unlikely that significant amounts of Cr203 could still be retained in the intestine after this time, the incomplete recovery of Cr2O3 could be due to 1) overestimation of intake, again unlikely in our experience, 2) minimal losses on toilet paper (15), 3) errors in analytical technique despite 100% recoveries ofknown amounts of Cr203 added to feces, or 4) some absorption of Cr by the intestine (15). Until the reasons for incomplete Cr203 recovery are known, and in light ofthe somewhat slower and more intermittent excretion of Cr2O3 compared to PEG, we prefer to use PEG in our balance experiments. In addition, Cr2O3 was more difficult to analyze, and was more difficult to administer than PEG.

HUMAN

NUTRITION

There was a significant correlation (P < 0.001) between the concentrations of nitrogen, calcium, phosphorus, magnesium, iron, and zinc relative to PEG in each fecal sample from all subjects in experiment I. This is evidence that PEG is evenly distributed in feces with respect to these nutrients, and that it is probably a valid marker for them. Wilkinson (2) also found constant ratios of calcium to PEG and phosphorus to PEG in feces. PEG appears to be a less suitable marker for sodium. Dietary sodium is almost completly abosrbed, and fecal sodium is mmimal except in illnesses such as diarrhea. It is therefore not surprising that fecal sodium does not correlate well with PEG. In general, those nutrients with the lowest percentage of absorption from the intestine, such as iron, zinc, and calcium, were most highly correlated with fecal PEG. We conclude that the use of PEG in this series of experiments enabled us to avoid some large errors in our balance studies, by identifying those subjects who were extremely slow to excrete the preexperimental diet, or who were relatively constipated for much of the study. When long-term, carefully monitored human nutrition studies are performed, the number of subjects is small relative to investment of time and money. The use of PEG permits the correction of data from those subjects with “nonideal” fecal flow rate. However, the fact that the average recovery of PEG was only 92.5% in our long-term experiments suggests that further work is needed before we can confidently use PEG as a fecal marker in humans. In these experiments, half of the subjects took more than a week to excrete food ingested before the study. While it is possible that the semipurified diets slowed transit time to some extent, it would appear that investigators who include balance data from the first 10 days of an experiment run a high risk of most fecal nutrients being derived from preexperimental dietary sources. Only by using a quantitative fecal marker, such as PEG, can this source of error be minimized. (]

way,

authors gratefully of the faculty

tional

Sciences,

including

Doris

University

Armstrong,

California

Marion

Baer,

at

Doris

I.

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Francoise

Frances Hess, and Donna

Costa,

Janet King, Spontak.

Cres Aban

Flores, Oddoye,

J. S. Marker perfusion techniques for intestinal absorption in man. Gastroen51: 1089. 1966. WILKINSON, R. Polyethylene glycol 4000 as a continuously administered non-absorbable fecal marker for metabolic balance studies in human subjects. Gut 12: 654, 1971. DAVIGNON, J., W. J. SIMMONDS AND E. H. AHRENS, JR. Usefulness of chromic oxide as an internal standard for balance studies in formula-fed patients and for assessment of colonic function. J. Clin. Invest. 47: 127, 1968. ALLEN, L., E. A. ODDOYE AND S. MARGEN. Proteininduced hypercalciuria; a longer term study. Am. J. Clin. Nutr. In press. MALAWER, S. J., AND D. W. POWELL. An improved turbidimetric analysis of polyethylene glycol using an emulsifier. Gastroenterology 53: 250, 1967. SMITH, R. H. Substances in the calfalimentary tract interfering in the determination of polyethylene glycol. Nature 182: 260, 1958. B0LIN, D. W., R. P. KING AND E. W. KLOSTERMAN. A simplified method for the determination of chromic oxide (Cr2O:,) when used as an index substance. Science 1 16: 634, 1952. MARGEN, S., AND J. C. KING. Effect oforal contraceptive agents on the metabolism of some trace minerals. Am. J. Clin. Nutr. 28: 392, 1975. HESS, F. M., J. C. KING AND S. MARGEN. Zinc excretion in young women on low zinc intakes and oral contraceptive agents. J. Nutr. 107: 16 10, 1977. KING, J. C., W. L. RAYNOLDS AND S. MARGEN. The absorption of stable isotopes of iron, copper and zinc during oral contraceptive use. Am. J. Clin. Nutr. 3 I: 1198, 1978. BAER, M. T., AND J. C. KING. Experimental zinc depletion in young men. Federation Proc. 37: 253, 1978. EATON, H. D., AND H. W. NORTON. Association between cerebrospinal fluid pressure and plasma vitamin A concentration of Holstein calves fed fixed intakes ofcarotene. J. Dairy Sci. 44: 1368, 1961. ROSE, G. A. Experiences with the use of interrupted carmine red and continuous chromium sesquioxide marking of human feces with reference to calcium, phosphorus and magnesium. Gut 5: 274, 1964. FIGUEROA, W. G., T. JORDAN AND S. H. BASSETF. Use of barium sulfate as an unabsorbable fecal marker. Am. J. Clin. Nutr. 21: 1239, 1968. SHARPE, S. J., AND M. F. ROBINSON. Intermittent FORDTRAN,

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oxide method of fecal marking in metabolic investigations on humans. J. Clin. Invest. 1960. 18. ALMY, T. P. The role offiber in the diet. In: Nutrition and Aging, edited by M. Winick. New York: John Wiley & Sons, 1976, p. 156. 19. Code of Federal Regulations no. 21, Food and Drugs. Washington, D.C.: United States Printing Office, 1977, section 172 . 820, p. 396. 20. SHIELDS, R., J. HARRIS AND M. W. DAVIES. Suitability of polyethylene glycol as a dilution indicator in the human colon. Gastroenterology 54: 331, 1968. 2 1 . BEEKEN, W. L. Clearance of circulating radiochromated albumin and erythrocytes by the gastrointestinal tract of normal subjects. Gastroenterology 52:

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use of trials