STUDIES
ON
THE
UTILIZATION
OF
HEXOSES
IN LIVER
GLYCOGEN
DISEASE
J. Fernandes, M.D., and J. H. van de Kamer, Ph.D.
The Children’s Department, Protestants Ziekenhui.g ‘s-Hertogenbosch, and the Central Institute for
Nutrition and Food Research T.N.O., Utrecht, The Netherlands
(Submitted April 15; revision accepted for publication September 29, 1964.)
ADDRESS: (J.H.v.d.K.) Central Institute for Nutrition & Food Research. T.N.O., 48, Utnechtseweg,
Zeist, The Netherlands.
470
PEDIATRICS, March 1965
I
N CHILDREN with liver glycogen disease the dietary carbohydrates demandspe-cia! attention. Rather than being yet an-other burden for an already overloaded
liver they should serve as a source of energy for the starving tissues. Therefore, it is of particular importance to know the metabolic
consequences of their inclusion in the diet
of these patients. The three main carbohy-drates are starch, sucrose, and lactose, com-posed of glucose, fructose, and galactose.
The fact that these hexoses differ in their metabolic 17 19 might have
conse-quences for their utilization by patients
with liver glycogen disease. To study this utilization, oral tolerance tests were per-formed on the basis of the following con-siderations:
As the glucose levels in the blood after
feeding a hexose alone will not give suf-ficient insight in its utilization, we decided to determine in addition the non-estenified
fatty acids (NEFA) and the lactate levels in the blood.
The NEFA level in the blood during a carbohydrate tolerance test is an indirect but very sensitive parameter for the
periph-era! utilization of the sugar used. These cir-culating free fatty acids, originating from the adipose tissue, decrease when carbo-hydrate is available to the tissue, but their level rises in the fasting ‘‘ or when the sugar absorbed cannot be utilized by
the peripheral tissues, NEFA then becom-ing a source of energy.
The simultaneous determination of the lactate levels may give some information
about carbohydrate breakdown. The data obtained should, however, be interpreted
with caution because an increase of this metabolite in the blood does not reveal whether its origin is dietary hexose or
gly-cogen in the liver.
Therefore, the experiments were planned as follows: oral tolerance tests with
glu-cose, fructose, or galactose were carried out in children with hepatic glycogen disease
(type I and type VI). During the test
glu-cose, total reducing substances, NEFA, and lactate in the blood were determined simul-taneously.
CLINICAL DATA
Four patients were studied, of which two had a phosphorylase deficiency and the
other two had a glucose-6-phosphatase de-ficiency. None of the children under study showed evidence of other abnormalities. No
medication was given. The pertinent clinical
and laboratory data are presented in Table
I.
METHODS
Blood glucose was estimated with
gin-cose oxidase in the reagent set TC-M of Boehringer.6
Total reducing capacity of the blood was estimated according to Folin and Wu.
Blood lactate was initially estimated chemically by a modification7 of the method of Long;8 in the course of the
in-vestigation this method was replaced by an
enzymatic method with the reagent set
TC-B of Boehringer.9 The results of both methods were identical.
0 We are much indebted to Dr. H. van Vals for
his valuable assistance in canning out these
TABLE I
PRINCIPAL DIAGNOSTIC FEATURES OF THE PATIENTS
Patient Age at Time of Study
Ilepa- Fasting Growth
tomeg- Ifypogly-
P*.etar-aly ceinia dation
Enzyme Defect Substrate investigated Level of the Deft-cient Enzyme Remarks 1. KB. O
6-7 + no yes Phosphorylase* Leucocytes I .5 mz
3. RR.
a
8 + moderate no Phosphorylase* Leucocytes 3. 6 m
2. 4 Ini.i
A second sibling
with the same
en-zyme defect was
not investigated
A.W.
9
16-19 ++ moderate yes
Glucose-6-phosphatasef
Liver (1 Xanthomata till 16 years
I.l. 9
‘2 + + severe yes Glucose-6-phosphatasef
Liver 0 A sister with the
same defect died
4.
* Method of Ilulsmann;4 normal values I 1-40 m moles glucose-i-phosphate formed per mg leucocyte protein
per iiiinute.
t Method of hers.5
Non-estenified fatty acids were estimated
according to Dole.bo Only 0.2 ml plasma was extracted instead of 1 ml as originally indicated by Dole. After addition of 0.8 ml of HO Dole’s procedure is followed ex-aetly. The titration is performed with 0.018
N
NaOH with an Agla micro syringe as a buret. It has to be stressed that the indicator solution must not be neutralized before use. The blank is of the same order of magnitudeas the titration figure for NEFA in normal plasma. The accuracy of the titration is, however, such that the reproducibility of
the determination is plus or minus 10% at a 60 pEq NEFA level per 100 ml. The figures in Table II may substantiate the modified procedure.
EXPERIMENTS
Glucose, fructose, and galactose were ad-ministered orally. The doses used were 2 g/kg body weight with a maximum of 50 g,
administered as a
10%
solution. During the test, capillary blood was taken at regularintervals for the determination of glucose, total reducing capacity, NEFA, and lactate (see the figures). The fasting period prior
between the tests being at least 3 days.
In Figure 1A there are some minor dif-ferences between the curves but they show the same pattern. Lactate rises in one pa-tient (K.B.) but fails to rise in the other pa-tient or in the control subject. The NEFA
shows a steady decline followed by a rise.
This decrease of the NEFA after oral
ad-ministration of glucose to normal subjects has been mentioned before.11
The NEFA curve clearly indicates the utilization of glucose not only by the liver
but also by peripheral tissues.
In Figure IB, the markedly elevated fast-ing levels show a steady decline which con-tinues during 3 to 4 hours. The
interpreta-TABLE II Plasma in ml H20 Added in ml Rotating the
Syringe in mm
A.W.
g4’cose
g/ovco.se mg/FOOml. /ctat9 mg/100
-NEICI4
,veq/io-Control
glucose
0 2 2 2P 3 35 40’i12 2)1 55Jhs.
/80
160
/40
/20
100
80
60
4’
KB. P.R. Control
4cose
$
,/,cos4,
glucose mg//O0 ml.
, - - -/dctate mg/ioo
---.-NEFA peq/’oo
-9/4’COSe
I
\
I,% -a’ o. O--O ...-o
*..,‘.,,,,,..,.!,,
)
‘ /
o- -o-’’ #{176} -o
‘ ._.‘_9’!
0 1 /2 213 35 0 1 12 25 350 ‘/2 1 1’2 2’,5 35I%’S
Fic. 1A. Glucose, lactate, and NEFA in blood following oral administration of
glu-cose to two patients with phosphorylase deficiency and one normal child.
tion of this drop in lactate level in contrast with the flat curve in normal subjects has
been discussed by other 3 14
NEFA shows a sharp drop. The durations of the tolerance test in A.W. seems to have
been too short to see a return of NEFA to fasting level. Evidently glucose is me-tabolized by these patients.
Figure 2A shows results of fructose
ad-ministered to 2 patients with phosphorylase
deficiency, and for comparison the data of
a normal child (control I). Fructose in the blood was not estimated directly but
cal-culated as the difference between total re-ducing capacity and blood glucose measured
by the glucose oxidase method. The identity
of this difference as fructose was not
checked and is therefore uncertain.
There is some increase of “fructose” in
the blood whereas the glucose level is con-stant or tends to decrease. The rise in lactate and fall in NEFA are more evident in patients KB. and R.R. than in control.
In each patient the result is of lesser de-gree than that after glucose in Figure 1A.
Thus fructose is utilized by these patients
though perhaps less well than glucose. Whether fructose is utilized directly or after its conversion to glucose is not de-terminable from the data presented.
In Figure 2B there is a small rise of total reducing capacity in patient I.L., the
cor-260
240
220
200
180 160 140 120 100
#{149}0
60 40 20
C
FIG. lB. Glucose, lactate, and NEFA in blood following oral administration of
KB. R.R. Control
friectose fructose fructose
i-.--. glucose mg/iooml
/‘eduCC4clmg//00
-- - - /actatemg/ioo
--- NEFA ,ue/ioo
-4
“
/‘‘
C ‘/2 1 /V2 2 2J 3 35 0 5 1 /J 2 25 S 31* 0 5 1 /V2 2 2Y2 .3 4h,-j
Fic. 2A. Glucose, reducing capacity, lactate, and NEFA in blood following oral
administration of fructose to two patients with phosphorylase deficiency and one normal child.
A.W.
f,wcfose
, - fWuc.c4. ,ng/o0i
glucose mg’/FOO
-lactate P7q//oo -,%‘EPA
,ueqj100-I.L.
I
I
‘-“
-
--:
Control
f,’wclose
0-- -.-- O’
-0 1 1; 2 2Va 5 354ka0 Va 1 2 2P 3#fr
Fic. 2B. Glucose, reducing capacity, lactate, and NEFA in blood following oral
administration of fructose to two patients with glucose-6-phosphatase deficiency and
one normal child.
0 ‘,‘s 1 15ff 2 2Yahr.r 200
/86
/6
/46
/26
/06
86
66
40
20
responding curve being flat in patient A.W.
I.L. has a persistent hypoglycemia. Both patients show a hyperlacticemia, lactate ap-proaching 200 mg/100 ml in patient I.L.
During the experiment this child showed an increasing hyperpnoe as a sign of acido-sis. The NEFA curves of the 2 patients show virtually no decrease in contrast with
the control.
It may be inferred from these data that
fructose does not become available to muscle and other peripheral tissues in any quantitatively significant amount.
Z60
240
220
200
180 160
140
120
/00
80 60
40
20
n
For Figure 3A, galactose in the blood was
not determined specifically but calculated
as the difference between total reducing
capacity and blood glucose measured by
the glucose oxidase method. The identity as galactose is open to the same doubt as was the ease with fructose.
There is an increase of total reducing ca-pacity with a simultaneous smaller rise of
the glucose level although the increase varies. Lactate and NEFA response follow-ing galactose administration in patients
FIG. 3B. Glucose, reducing capacity, lactate, and NEFA in blood following oral
administration of galactose to two patients with glucose-6-phosphatase deficiency and
one normal child.
/80
/60
/40
/20
IX
80
6
4’
20
KB. P.R. Control
gelactose
J#{188}
I
cvi
I’,I I‘, I
1’
x,
#{244}’go/octose ge/octose
- g/wcosemg/iooml. 4
- eedwc.ca/.’.mg/ioo
---- lactate mg/ioo
-“
--- NEic4,sieq/ioo
-q
::;z:::::=;;
-o.-- #‘ p.,/ ‘0
1
00 /2 1 l5fr2 2Y .5 35 0 ‘/2 1 /V2 2 2 3 3’4 0 ‘/ 1 P12 2 2’/z 3 3i 4 hrs.
FIG. 3A. Glucose, reducing capacity, lactate, and NEFA in blood following oral
administration of galactose to two patients with phosphorylase deficiency and one normal child.
after fructose administration (Fig. 2A). Fur-thermore the increase in lactate in patients with phosphorylase deficiency tends to be more pronounced than the nearly constant
level in the normal child.
Thus galactose is utilized by these pa-tients, either directly or indirectly after its conversion to glucose in the liver.
The curves of total reducing capacity in Figure 3B are dissimilar: a constant level in patient A.W., a marked increase in
pa-tient I.L. with a concomitant decrease of glucose to hypoglycemic values. Consider-ing the constant high NEFA level in both
patients, there is no evidence of substantial
utilization of this hexose by the peripheral
tissues. Lactate shows an increase. Clinical signs of acidosis, however, were not
ap-parent.
The pattern of metabolic events follow-ing the administration of fructose and galactose to patients with glucose-6-phos-phatase deficiency thus is strikingly differ-ent from the glucose effect.
In order to compare fructose and
glucose itctose
H
260
240 220
200
180
160
140
120
100
80
60
40
20
,,‘b- /
..
01 234 56 788/OMsOl 254 #{231}6 789*,’.’.
A B
FIG. 4A and 4B. Glucose, reducing capacity, lactate, and NEFA in blood following
oral a(lnhinistration of glucose, fructose, glucose respectively (Fig. 4A) and of glucose, galactosc, glucose respectively (Fig. 4B) to a patient with glucose-fi-phosphatase
deficiency.
I.L.
- reO’uc.C4f. mg/Foos7/
glucose ng/F00
-/ectote mg//CO
-,%/ic#CA
,veq/’oo-glucose glucose go’lacEO.Se g/uC0s
N
During 10 hours the two hexoses were
administered orally to patient I.L. (glucose-6-phosphatase deficiency). The doses used
were 1.5 g/kg as a 30% solution, given
hourly. The sequence of administration was
as follows: glucose 3X, fructose 4X,
fol-lowed again by glucose 3 X . This A-B-A
de-sign was carried out in the way described in order to attain a metabolic steady state at the end of each period. Each blood sample
was taken immediately before administer-ing next sugar dose.
In Figure 4A, the curves of glucose and total reducing capacity coincide during both
glucose periods. They diverge somewhat during the intervening fructose period.
Both lactate and NEFA levels fall during each glucose period but increase during the
fructose period.
The rapid rise of lactate following the
first fructose feeding is indirect proof of
the absorption and degradation of this
sugar. During the entire fructose period the blood glucose level declines steadily until it has dropped to the alarmingly low value of
8 mg/100 ml. In agreement with these values the child became increasingly
hy-poglycernic and acidotic. Recovery from this state of metabolic disturbance was slow in spite of appropriate treatment with
glu-cose.
The test whose results are shown in
Fig-ure 4B was carried out in the same way with the same patient except that blood
as-say in the preliminary fasting period was omitted. In this experiment the sequence was : glucose 3 X , galactose 3 X ,glucose 3 X.
Again the curves of glucose and total re-ducing capacity coincide during both
glit-cose periods, to diverge distinctly during
the intervening galactose period. Lactate rises during the galactose period and at the
same time there is a minor increase of
NEFA. The rapid rise of lactate following
the first galactose administration is evi-dence of the immediate degradation of this
sugar.
COMMENT
It is well known that the metabolism of
In children with glucose-6-phosphatase liver, their direct utilization as such by
pe-ripheral tissues probably being of minor
rt71#{176} The last step in this trans-formation of fructose and galactose to glu-cose is the dephosphorylation of glucose-6-phosphate. In complete agreement with this, patients with glucose-6-phosphatase
deficiency react quite differently from nor-ma! children on the administration of frue-tose and galactose, because the absence of
this enzyme blocks the transformation of these two hexoses into glucose.15 In these
patients, therefore, glucose alone can counteract the effects of fasting : hyper-lacticemia and high NEFA levels.16
Fruc-tose and galactose do not alleviate the phe-nomena of the fasting state. The almost
im-mediate rise of blood lactate following the
administration of fructose or galactose sug-gests conversion of these sugars to lactic acid. Feeding fructose and galactose to pa-tients with glucose-6-phosphatase deficiency
therefore has an unfavorable effect on both their fasting hypoglycemia and their
acido-sis.
When phosphorylase is defective the dif-ference between the utilization of glucose
on the one hand and fructose and galactose on the other hand is less striking. This is
easily understood as the transformation of these latter two sugars to glucose remains possible in this case. The pattern in these
patients is hardly different from that of a
normal child. Yet even in these children glucose seems to be more favorable in its utilization by liver and other tissues as measured by NEFA and lactate levels in
the blood.
From our experiments the sequence of events after the administration of glucose,
fructose and galactose respectively seems to be as follows:
Glucose, after absorption, is partially
stored or metabolized in the liver, the rest is utilized by muscle and other peripheral
tissues.
Fructose is almost exclusively utilized by
the 718 J the liver fructose is either
degraded or converted to glucose-phosphate in a rather complicated way and then may
be deposited as glycogen, the latter process probably being the more important.18 Fol-lowing these metabolic processes glucose
may be released by the liver and metab-olized by the other tissues. Fructose as such
has no or minimal nutritional value for peripheral tissues.
In our patients fructose seems to lead to
fairly strong lactate production, probably in the liver.
Galactose appears in some patients in the
blood as an appreciable amount of non-glucose-reducing substance. Our data do
not show evidence of substantial utilization in the peripheral tissues. It must be
con-eluded that the liver represents the main site of metabolism of this hexose.19
Consid-erable lactic acid production is observed in our patients following galactose
adminis-tration.
Furthermore our experiments indicate that fructose and galactose have a very
un-favorable effect on patients with glucose-6-phosphatase deficiency and therefore should be excluded from their diet. Also, in
pa-tients with a phosphorylase deficiency these sugars are less favorable than glucose, but
there seems to be no objection against in-eluding these two sugars in the diet of these
patients.
SUMMARY
The utilization of glucose, fructose, and
galactose was studied by oral tolerance tests in four patients with liver glycogen disease, two having a phosphorylase de-ficiency, the other two a glueose-6-phos-phatase deficiency.
The utilization of these sugars was
evalu-ated by the estimation of glucose, total reducing capacity, non-esterified fatty acids (NEFA), and lactate in the blood. The data were plotted against time and the curves obtained in the two types of abnormal
car-bohydrate metabolism and in two control
children were compared.
The curves in children with phosphory-lase deficiency approached the normal
deficiency, however, the curves for fructose
and galactose were highly abnormal.
It was concluded that in the latter case fructose and galactose should be excluded
from the diet.
REFERENCES
1. Karlson, P. : Introduction to Modem
Biochem-istry, 1963.
2. Goodman, D. S., and Gordon, R. S. : The
metabolism of plasma unesterified fatty acid.
Amer. J. Clin. Nutr., 6:669, 1958.
3. Gordon, R. S., Cherkes, A., and Gates, H.:
Unesterified fatty acid in human blood
plasma. II. The transport function of
un-esterified fatty acid.
J.
Clin. Invest., 36:810,1957.
4. H#{252}lsniann, W. C., Oei, T. L., and Creveld, S.
van : Phosphorylase activity in leucocytes
from patients with glycogen-storage disease.
Lancet, 11:581, 1961.
5. Hers, H. G. : Etudes enzymatiques sur
frag-ments h#{233}patiques; application
a
laclassifi-cation des glycog#{233}noses. Revue Int. Hepat.,
9:35, 1959.
6. Hugret, A.St.G., and Dixon, D. A. : Enzymic determination of blood glucose. Biochem.
1.’
66:12, 1957.7. Weijers, H. A., and Kamer, J. H. van de:
Diarrhoea caused by deficiency of
sugar-splitting enzymes. II. Acta Paediat.
(Stock-holm), 51:371, 1962.
8. Long, C. : The stabilisation and estimation of
lactic acid in blood samples. Biochem. J.,
40:27, 1946.
9. Scholz, R., Schmitz, H., B#{252}cher, Th., and
Lampen, J. 0. : Ueber die Wirkung von
Nystatin auf B#{228}ckerhefe. Biochem. Z.,
331:71, 1959.
10. Dole, V. P., and Meinertz, H. :
Microdeter-mination of long-chain fatty acids in plasma
and tissues. J. Biol. Chem., 235:2595, 1960.
11. Gordon, R. S., and Cherkes, A. Unesterified fatty acid in human blood plasma. J. Clin.
Invest., 35:206, 1956.
12. Lowe, C. U., Sokal, J. E., Mosovich, L. L.,
Sarcione, E. J., and Doray, B. H. : Studies
in liver glycogen disease. Amer. J. Med.,
33:4, 1962.
13. Howell, R. R., Ashton, D. M., and
Wijngaar-den, J. B. : Glucose-6-phosphatase deficiency
glycogen storage disease. Studies on the
interrelationships of carbohydrate, lipid, and
purine abnormalities. Pauirmcs, 29:553,
1962.
14. Sokal, J. E., Lowe Ch.U., Sarcione, E. J., Mosovich, L. L., and Doray, B. H. Studies
on glycogen metabolism in liver glycogen
disease (von Gierke’s disease). J. Clin.
In-vest., 40:364, 1961.
15. Mason, H. H., and Andersen, D. H. : Glycogen
disease of the liver (von Gierke’s disease)
with hepatomata; case report with metabolic
studies. PEDIATRICS, 16:785, 1955.
16. Benedetti, A., Nardini, A., and Picone, C.:
Syndrome dii
a
l’absence cong#{233}nitale deglucose-6-phosphatase chez deux adultes
fr#{232}reet soeur. Ann. Endocr., 22:647, 1961.
17. Renold, A. E., and Thorn, C. W. : Clinical usefulness of fructose. Amer. J. Med.,
19:163 1955
18. Leuthardt, F. : Der Stoffwechsel der Fructose.
Schweiz. Med. Wsch., 90:455, 487, 1960.
19. Schwartz, R., Ashmore, J., and Renold, A. E.:
Galactose tolerance in glycogen storage
dis-ease. PEDIATRICS, 19:585, 1957.
Acknowledgments
We are indebted to Professor H. Hers, Louvain,
and Dr. F. Huijing, Amsterdam, who kindly
carried out the enzyme assays, and to Drs. Th. M.
van der Kley, J. van Marle, and L. H. J.
Ra-maekers, through whose courtesy and co-operation
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