ASSESSMENT
OF
THREE
METHODS
FOR
MEASURING
INTESTINAL
FAT
ABSORPTION
IN INFANTS
AND
CHILDREN
Richard B. Goldbloom, M.D., and Roy M. Blake, M.D.
With the technical assistance of Dorothy Cameron, R.T.
Department of Paediatrics, McGill University, and the Department of Metabolism,
The Montreal Children’s Hospital
(Submitted June 15; accepted for publication July 28, 1964.)
This vork was su1)ported by Grant No. MA-1387, Medical Research Council of Canada.
ADDRESS: (R.B.C.) The Montreal Children’s Hospital, 2:300 Tupper Street, Montreal, P.Q., Canada.
PEDIATRICS, December 1964
M
OST available methods for measuring intestinal fat absorption have a com-mon limitation: tile results depend ondi-gestion as well as on absorption of tile
ingested lipid. Several of these techniques have other practical disadvantages when
ap-plied to infants and children: The I1
triolein test requires at least three days to complete, and loss of a single stool speci-men invalidates the result. The P’-labeled oleic acid test is useful in distinguishing impaired fat digestion, hut is equally time-consuming. The reliability of the Vitamin A ai)sorption test has been seriously ques-tioned.1 There is considerable variability
111 the recommended dosage and in the
in-terpretation of what constitutes an
ab-normal response. The modified lipiodol
ab-sorption test,’ although relatively simple,
takes 12 to 18 hours to perform; and de-pendabie results require more careful con-trol of the assay technique than was origi-nally recommended. \Ieasurement of the increase in serum turl)idity (lactescence) after administration of a test (lose of butter-fat has certain theoretical and practical attractions : First, tile test substance is a inajor component of normal (hetary fat in chui(lren. Second, urine or stool collections are not required, since the test is per-formed on microsamples of capillary blood. Finaliv, only three or four hours are neecle(l to complete the test.
Analysis of the fecal fat content on a
measured intake over a 4-day period is probably the most exact index of fat ab-sorption. Tile results undoubtedly reflect a c:miplex of interdependent processes,
in-eluding digestion , absorption, secretion,
and synthesis. In children, the technique is
only practical and accurate if carried out in
a metabolic unit, with special laboratory
facilities.
In this study, using 4-day fecal fat ex-cretion and fat balance measurements as the reference standards, we have compared the reliability of three other measures of in-testinal fat absorption in infants and chil-dren: Vitamin A, U31-triolein, and butterfat absorption as determined by the rise in
serum turbidity.
PATIENT MATERIAL
Twenty-two infants and children,
rang-ing in age from 4 months to 13 years, were
admitted to the Metabolic Unit of The Montreal Children’s Hospital for about 2
weeks eacil. More than half of these
chil-dren were known to have intestinal absorp-tive defects, and the remainder had various forms of growth failure. The diagnostic composition of the group is indicated in Table I. In three of the four children with
gluten enteropathy. the diagnosis was
sub-stantiated by peroral intestinal biopsy,#{176}
and in all by their subsequent response to a gluten-free diet. For technical reasons, Vitamin A and triolein absorption tests could not he carried out in three children.
Fat Balance
METHODS
Each patient was observed for 3 days by the dietitian to estimate the average
815
TABLE I
l)I.GNosTIc ColleosiTIoN OF THE OF
PATIENTS STUDIED
. . NO. of
I)iagnoszs Patient.
(;Iuteii enteropathy 4
(;iardiasis 3
Structural abnormalities of t lie intest ine*
Cystic fibrosis of the pancreas Familial SIllilli stature llvpocaloric nialnutrition
Rheumatoid arthritis
Indiagnose(l “failure to thrive’
Total 22
* The category “structural al)nornialities of the
in-testine” includes one child with extensive surgical
bowel resection, one postoperative Ilirschsprungs
disease with chronic diarrhea, and one cIiil(l with a
gastrojejunostoiny and “dumping syndrome.
daily fat intake and to determine food pref-erences. A balance diet, with fat content approximating the child’s usual daily in-take was then instituted. After a 2-day period of equilibration, all stools were col-lected between two carmine markers, given
exactly 4 days apart. Total fecal fat was
(letermined by the method of Van de Kamer, modified as follows:
1. The 4-day collection of stool was homogenized in a commercial Waring Blendor, and analyses were performed in duplicate on 10 ml aliquots. (Mean van-ation in duplicate analyses was zero; maxi-mum range of difference ±1.7%.)
2. Toluene was used for lipid extraction as recommended by Jover and Gordon.7
3. Results were expressed both as grams of fecal fat/day and as the percentage of fat intake excreted during the 4-day test period.
In 19 of the studies, sample daily diets were homogenized and aliquots analyzed
for fat content. Tile results were tllen
com-pared to the calculated fat content of the diets.
U ‘-TRIoLEIN ABSORPTION was measured
by a modification of the method of Spector
and his associates.” All children were given
Lugol’s solution, 5 drops bid. for 1 day
prior to the test and on the first test day, to prevent thyroid uptake of the adminis-tered isotope. P’-triolein#{176} was given in a dose of 8-10 .c in 30 ml of a lipid emul-sionf whicil contained 20 gm of corn oil. All feces were collected thereafter, until
radioactivity had disappeared.
VITA1’SIIN A ABSORPTION: This was
deter-3 mined according to the method of Besse’ and associates.’ Vitamin A was omitted
:
from the diet for 24 hours prior to the test.The test dose was 0.2 ml of oleum
perco-5 morph (6,00() U.S.P. units) per kilo of body
weight.
BUTTERFAT ABSORPTION was determined
by the micro-modification of the method of Osmon and associates.’#{176} The patient was
fasted from midnight, and at approximately
9:00 A.M. a test dose of 0.5 gm butterfat/k was given as 15% cream. Capillary blood specimens of 0.1 ml each were obtained in the fasting state and at ilourly intervals up to 4 hours. Optical density of the serum
was read at 620 m:j. in a Beckman-DU Spectrophotometer, using micro-cuvettes with a 1 cm light path, and a pinhole adapter.
\Vith this technique, maxilnum serum
tur-bidity always occurred at 3 hours. In our experience a normal response is repre-sented by an increase in serum turbidity of more than .170 O.D. between the fasting and 3-hour specimens. In 64 normal mdi-viduals of all ages (1 month to adult), the
mean rise in O.D. at 3 ilours was .185 ±
.006 S.D. The range of responses in this group of normal subjects is illustrated in Figure 1.
RESU LTS
Sample daily intakes of 19 of the 22 hal-ance diets were analyzed for fat content. The results were compared to the calcu-lated fat content of the respective diets. As shown in Table II, excellent agreement was obtained between the analyses amid the calculated estimates.
Supplied by Charles E. Frosst Co. Ltd., and
stated to be 90% pure triolein.
SERUM TURBIDITY RESPONSE to BUTTERFAT ABSORPTION in$4 NORMAL INDIVIDUALS
(M.an± Rang.)
SERUM TURBIDITY O.D.
16
14 12
10 FECAL
FAT
GM/DAY 6 7.
7
HOURS
‘FABLE II
IN
4HOUR 400
RISE IN
SERUM VITAMIN A
lU.
$00
1000
1200
Fir.. 1. The response of serum turbidity (optical
(lUIlSitV) to administration of butterfat (0.5 gm/k) as 15% creani in norn1al individuals. N1axiniun
turbidity as always observed at 3 hours.
Values obtained for the fecal fat output
(gm/day
)
correlated closely with fecai fatexpressed as a percentage of the intake (Fig. 2). The relative contribution of dietary fat
to total fecai lipids is not clearly
estab-iisiled, eitiler in normal or in malahsorptive
states. Therefore, to evaluate the three
methods as fairly as possible, statistical
coml)anisons were made witil both
expres-Si0115 of fecal fat excretion.
Tile results of tile Vitamin A absorption test faiied to correlate with the percent-age of the fat intake excreted during the 4-day test I)eriOI (Fig. 3). There was a similar lack of correlation with fecal fat
output, expressed as gm/day (r = 0.37,
p > 0.1).
(AI(uI.ATED VS. ANAlYZED FAT CONTENT OF 1)1Km
(SKI) FOR BALANCE STUDIES
No. of l)iets Analyzed 19
Range of }at (‘ontent/I)ay
(Calculated) 40-(O gui.
Mean 1)itierence ±5.1). -0 .373 ±-2 .5’2 gui.
((‘akulated-Auialvzed) (P>O.5)
fluenuige of fat (oultent/day refers to the variation iiidaily intake between different children included in
the study. Iii each individual case, daily intake wa
unaintaiuue(l vithuiui fairly narrow limits.
y= 338+O207x
r= 070 p< .001
10 20 30 40 50 60 FECALFAT IINTAKE
II(;. 2. Comparison of the two expressions of fecal
fat balance-quantitative (laily excretion ilI1(l total 4-d:i’ excretion (XI)u(SS((l as a percentage of the intake. A fairly close relationship was oljserved in
this group of pltients.
Triolein excretion correlated with the percentage of fat intake excreted in the feces at the 5% level (Fig. 4a); a better
reia-tion was observed when the results were
Pltte(l against the (11antitative daily fat
excretion (Fig. 4h).
The maximumii (3 hour) rise in serum
tur-hiditv following adniinistration of butterfat correlated well with both parameters of fecal fat output (Figs. 5a and b).
COMMENT
In assessing tile significance of these
re-suIts, tile first premise to be questioned is
0 35 P02
10 20 30 40 50 60
FECAL FAT AS ‘/, INTAKE
Fir.. 3. Comparison of thU 4-hour rise in serum
Vitamin A levels with fecal fat excretion expressed
as a percentage of the intake over a 4-day perio(l.
80
70
60 50 ECAL TRIOLEIN 40
EXCRETED 30 20 10
y O1720 0007x r- -0813 pnO 001
#{149}NNNN
y67l 40902x
r=0461 p0OS
180 1 70
160 .150 .140
.130
180
170
160
.15c
140
10 20 30 40 50 60
FECAL FATh INTAKE
y.t 174-00019* ,-0675
4 0001
PEAK
SERUM
TURBIDITY 0. D.
....
.. . -: -. -S...
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20
10
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p< 0001 10 20 30 40 50 60 70
FECAL FATX INTAKE
Fmc. 4a. Fecal excretion of I3-triolein, compared
to the percentage of nicasured fat intake excrete(l
OVU t 4-day 1)triod.
the reliability of tile reference standards. Despite consideral)le study, few
investiga-tors are fully agreed on tile proportionate
origins of fecal fat: Normal adults have
been siiwn to excrete reduced but sig-nificant amounts of fat while receiving es-sentially fat-free diets.11 Studies by Wol-laeger and associates suggest that fecal fat content is moderately augmented by increas-ing dietary intake.’ Holt and co-workers
found that reducing the fat intake of
in-fants to 1 gm/day diminisiled fecai fat
out-put to 0.5 gm/day.’ By contrast,
Anne-gers14 found little variation in fecal fat
out-put vben the daily intake of normal adults
80
70
60 50 FECAL TRIOLEIN 40
x
EXCRETED 30
.2
2 4 6 8 10 12 14 16
FECAL FAT GM./ DAY
Fic. 4b. The quantitative daily fecal fat content
(luring the same period.
PEAK
SERUM TURBI DITY
0.D.
2 4 6 8 10 12 14 16
FECAL FAT GM./DAY
Fic. 5. Com1mrison of peak serum turbidity
fol-loving adiiiinistration of 1)utterfat (0.5 gni/k bod
‘eight) to the tvo 1)arauileters of fetal fat excretion.
was varied between 93 and 150 gIn.
In patients with sprue, the avaiiabk’
evidence indicates that the daily quamitity
of fecal fat is largely, though not
coii-pletelv, dependent on tile anlount in-gested.
The endogenous component of fecal fat
may derive in varying amounts from lipid excretion , bacterial synthes is, (les(IIIclfllate(l
mucosal cells and bile. Nevertheless, the
measurement of fecal fat excretion, either in total or in balance terms, seems to reflect the Iresemlce and severity of intestinal
rnalabsorption with fair accuracy, and is
the generally accepted standard of
refer-ence. :
The over-all 1)rohlem was sunlmarized
almost ten years ago by Frazer’7 and his
cOmmellts do not appear to require
modi-fication in the light of subsequent
investi-gations: “It is difficult, at tile present time,
to define with any precision the relative
gun. ingested fat
that go to form faecal fat in any individual
case of steatorrhea; it is likely that this
proportion iiia’ vary from case to case, and, indeed, it may also vary from time to time
in tile same case.”
\Ve chose to compare the results of tile three aI)sorption tests to both expressions
of fat excretion (gm/day and % intake). The
use of 1)0th standards did not alter the
as-sessment of the results of the Vitamin A or
of tile serum turbidity tests. As will be
seen, the significance of tile triolein results
depended on which of the two standards
was applied.
Measurement of Vitamin A absorption,
as performed here, was unreliable as an
index of either normalcy or of impaired in-testinal fat al)sorption. This confirms tile
findings of two earlier studies: Weijers and Van de Kamer’ found no consistent rela-tionship between iml)airment of Vitamin A absorption and the coefficient of fat ab-sorption.#{176} More recently, coin-pared Vitamin A absorption to fat balance measurements in 87 patients. He obtained an abnormal response to one test associated
with a normal response to the other in 48%
of cases. Fasting Vitamin A levels failed to
correlate with the results of subsequent
tolerance tests.
The extent to which the U’-tniolein test
reflects intestinal fat absorption has been
assessed ni several previous investigations,
vith variable results: Cox” studied 100
subjects, 33 of whom had steatorrhea, as cletermine(1 by a (laily fecal fat of 7 gm or
over. He found that tile triolein test gave
normal values in normal subjects, hut failed to confirm tile steatorrhea in more than half the patients with increased fecal fat. Pimparkar and co-workers#{176}’ found a 15% incidence of false negative results in apply-ing the fecal tniolein excretion test to 30 patients with idiopathic steatorrhea or pan-creatic disease. Among individuals with
* gin. iuogested fat -gIn. excreted fat
X1($).
normal fecal fat excretion, triolein
excre-tion was elevated in al)out 10%.
Davis and Badenoch obtained good
cor-relation between radiotriolein tests and
chemical fat determinations in 9 patients vith steatorrhea. Hetenvi and
O’Sulli-van2 found a good correlation between
fecal P’-trioiein excretion and fecal fat
excretioll ill 59 individuals. However,
ex-amination of the data suggests that the
de-gree of correlation may have been weighted
l)y a large number of normal and
func-tional diarrhea cases.
Several potential sources of error in tile
I1tniolein test have been recognized.
Commercial triolein preparations may vary
considerably iii purity, and this factor may
he an important determinant of test
re-71 Since the urine contains significant
quantities of the radioactive label, espe-cially in the first 24 hours after isotope ad-ministration, any contamination of tile fecal collection will lead to falsely high excretion (low absorption) values. The possibility of such contamination is very real in infants and children. Iisleading results and
exas-peration also follow the loss of any part of
the stool collection.
The relative merit of the triolein test in
tiliS study is difficult to assess because of
the difference iii degree of correlation
oh-tamed \Vitil the two standards of fecal fat
excretion. A close relationship with
quan-titative daily fecal fat was found, but the
test results were less closely related to fecal
fat expressed as a percentage of intake. It
has already 1)een pointed out that present knowledge does not permit a clear choice between these two reference standards.
Tile results of tile serum turbidity test
silowed a close correlation with both
meas-tires of fecal fat output. For reasons which
are not entirely clear, this method has
re-ceived little attention or careful evaluation
since first described by Osmon and
associ-ates)#{176} The observations which form the
basis of the test are as follows: Tile major
portion of absorbed fat is in tile form of neutral fat, which appears in the serum as
Aibrink and her associates27 showed that
the increased turbidity of blood serum
which followed ingestion of fat was dime to the increase in neutral fat content. Tile
spectropilotometnic measurement of serum
turbidity or lactescence is, in effect, a
sim-pie equivalent of chylomicron counting.
Os-mon et a!.’” found that tile rise in serum
turl)idity correlated well with increases in
total serum lipids and with chylomicron
counts. Gardner and Perez-Santiago28
con-firmed these observations, and stated that
the test was more reproducible than
chylo-micron counting.
The method is easily adapted to avail-able spectrophotometnic equipment. As
originally reported, tile results were
ex-I)ressel in “Klett Units.” \‘Ve found it
sim-pier to record the optical density readings.
The chief theoretical disadvantage to tile
method (aside from those shared by other fat absorption techniques), is the possible
effect of clearing of chylomicrons
(beta-lipoprotein lipase activity) on the serum turbidity curve. The importance of this factor has not been fully assessed. How-ever, the range of normal value is reason-ably narrow (Fig. 1); and we have found a
high
Or(ler of reproducibility with repeattesting in both normal and steatorrheic
in-dividuals. Tile close relationship with fecal
fat excretion also suggests that the
clear-ing factor is not of major importance.
Stool and urine collections are anathema
to many hospital personnel in wards and
laboratories not specially staffed and equipped to collect and analyse them ac-curateiy. However, lack of dependence on
excreta is not tile sole virtue of tile
butter-fat absorption test. The short time involved
and the use of natural dietary fat in a
palatable form and a physiological dose
are added attractions, particularly in tile
pediatric age group.
It is unlikely that any present or future
test will detect every instance of
impair-ment in the complex of transport processes
we loosely call absorption. If one is aware
of the numerous limitations affecting tile
measurement of intestinal absorption, the
importance of in(Iividualized assessment of every patient is readily appreciated.
SUMMARY
Intestinal fat al)sorption has been meas-tired in 22 infants and children with
van-oils forms of steatorrhea or growth failure,
using four different techniques. Vitamin A
absorption, ‘‘triolein aI)sorption ,
butter-fat absorption (as measured by alteration
in serum turbidity), and 4-day fecal fat hal-ance. The last of these was used as the reference standard, expressed both as gm/ day and as percentage of intake excreted
over tile 4-day period.
The measurement of Vitamin A
absorp-tion was unreliable as an index of tile
in-testinal handling of lipid. I’’-tniolein
at)-sorption correlated vell with fecal fat output, expressed as gm/day. A close rela-tionship both to fecai fat balance and to
(Juantitative (laily excretion was attained
by results of tile serum turbidity metilod for butterfat absorption. The simplicity and special advantages of this technique are described.
REFERENCES
1. \Veijers, 11. A., and Van de Kamer, j. II.: Coeliac (liseaSe : I. Criticism of the various
uiiethods of ioivestigation. Acta. 1ted. , 42 : 24,
1953.
2. Ilillman. R. \V., and Becker, N. II. :
Absorp-tion of ingeste(1 \7itamin A.
Gastroenterol-ogv, 32:738, 1957.
3. Silverman, F. N., and Shirkev, 11. C. Fat ab_
sorption test using iodized oil, with I)lrticuu_
lar application as a screening test in the
(hag-nosis of fihrocvstic (lisease of the l)lncreas.
PEDIATRICS, 15: 14:3, 1935.
4. jones, \V. 0., and (Ii Sant’Agnese, P. A.:
Laboratory aids in the (liagnosis of nial-absorption in I)eli1tricS : Lipiodol absorption
as a simple test for steatorrhea. J. Pediatr.,
62:44, 1963.
5. O’Brien, D., \Valker, I). M.. and Ibhott, F. A.:
S!ecificity of the iodized oil test for fat absorption. (Letters to the Editor) PEI)!-ATRICS, 23:422, 1959.
6. Van de Kamer, j. II., ten Bokkel 1-luinink, 11., and %Veijers, H. A.: Rapid method for the
determination of fat in feces. J. Biol. Chem.,
177:347, 1949.
820
(jt1111titatiVe analysis of feces with special
reference to fatty acids. J. Lab. Clin. Med.,
59:878, 1962.
S. Spector, S.. Matthews, I,.. V. Leuiini, F. i. Van Erp. Y., and Cline, J.: Study of fat
al)sorptn)n utilizing I:Ilihelecl corn oil in infants and children with and without
steatorrhea. PEDIATRICS, 22 : 515, 1958.
9. Bessey, 1). A., Lowry, 0. H., Brock, M. J., and
Lopez, J. A. The determination of Vitamin
A and carotene in sn)all 9uantities of 1)100(1
s(runl. J. Biol. Chem., 166: 177, 1946.
10. Osmon, K., Zinn, W. J., an(1 Wharton, C. V.:
Simplified test of fat absorption. j.A.M.A.,
164:633, 1957.
11. Lewis, C. T.. and Partin, MS.: Fecal fat on
an essentially fat-free diet, J. Lab. Clin.
Me(l., 44:91, 1954.
12. \Vollaeger, E. E., Comfort, \I. W., and
Oster-berg, A. E. : Total solids, fat and nitrogen in the feces. III. A study of normal persons taking a test (liLt containing a moderate amount of fat; comparison with results
oh-tuned with normal persons taking a test diet Containing a large amount of fat. Castroen-terology, 9:272, 1947.
13. Holt, L. E., Jr., Tidwell, H. C., Kirk, C. M.,
Cross, D. M., and Neale, S. : Studies in fat
nietabolism. I. Fat absorption in nonl)al in-fants. J. Pediat., 6:427, 1935.
14. Annegers, J. H. : Function of pancreatic juice
and of bile in assimilation of dietary
tn-glyceride. Arch.
mt.
Med., 93:9, 1954.15. Asenjo, C. F., Rodriguez, M. R., and Cancio, NI. : 1)iet 017(1 fecal fat excretion in sprue.
Fed. Proc., 16::380, 1957.
16. Comfort, M. W., Wollaegen, E. E., Taylor, A. B., and Power, M. H.: Nontropical sprue.
Observations on absorption and metabolism.
Gastroenterologv. 23:155, 1955.
17. Frazer, A. C.: Steatornhea. Bnit. Med. J., 2:805,
1955.
18. Wormsley, K. C.: Use of labelled tniolein, Vitamin A, and D-xvlose in the diagnosis of
malahsorption. Cut, 4:261, 1963.
19. Cox, A. C. : Assessment of the radiotniolein test
in steatonrhea. Bnit. Med. J., 2:933, 1961. 20. Pimparkar, B. D., Tulsk , E. C., Kaiser, M. H..
an(l Bockus, H. L. : Correlation of radioactive and chenlical fecal fat deterullinations in the
malabsorption syndrome. I. Studies in normal
man and in functional (lisor(lers of the
gas-trointestinal tract. Amer. J. Med., 30:910.
1961.
21. Pimparkan, B. D., Tulskv, E. C., Kaiser, M. F!.,
and Bockus, H. L. : II. Results in i(liol)atlliC
steatorrhea and (liseaSes of the pancre:1s.
Amer. j. Me(1., 30:927, 1961.
22. Davis, A. E., and Badenoch, j. : Radio-oleic
acid and rada)tniolein tests in differential
di-agnosis of steatorrhea. Bnit. Med. J., 2:638,
1962.
23. Hetenyi, C., and O’Sullivan, P. M. : A
compani-SOIl of laboratories tests in the malabsorption syndrome. Can. MCCI. Ass. J., 90:116, 1964.
24. Tuna, N., Mangold, 11. K., and v1osser, D. C.
Re-evaluation of the I’3-tniolein test. J. Lab. Clin. Med., 61:620, 1963.
25. Frazer, A. C., and Stewart, H. C. :
Ultramicro-scopic particles in normal human blood. J.
Physiol., 90: 18, 1937.
26. Elkes, J. J., Frazen, A. C., and Stewart, H. C.:
Conlposition of particles seen in normal
human blood under dark-ground
illuinina-tion. j. Physiol., 95:68, 1939.
27. Alhrink, M. j., Man, E. R., and Peter, J. P.:
Relation of neutral fat to lactescence of
serum. J. Clin. Invest., 34:147, 1955. 28. Gardner, F. F!., and Perez-Santiago, E. : Oral
absorption tolerance tests in tropical sprue. Arch.
mt.
Med., 98:467, 1958.Acknowledgments
The authors are grateful to l)r. I)onald K. Clogg
for performance of the I’31-triok’in tests; to Mrs. Diane Cox for assistance with statisical analysis of the data; and to Miss Sandra Pilkington, RN., Nurse-in-charge, Metabolic Unit, The Montreal
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