BIOCHIMICA ET BIOPHYSICA ACTA 327

BBA 26453

GALACTOSE TOXICITY IN THE CHICK: OXIDATION OF

RADIOACTIVE GALACTOSE

HENRY J. WELLS, MAUREEN GORDON AI~D STANTON SEGAL

Division of Biochemical Development and Molecular Diseases, Children's Hospirtal of Philadelphia and Department of Pediatrics, University of Pennsylvania Medical School, Philadelphia, Pa. (U.S.A .)

(Received June Isth, 197 o)

SUMMARY

Radioactive galactose has been shown to be converted to 14C0~ by both the intact chick and by various tissues in vitro. The pattern of 14C02 liberation by the in- tact chick was not related to the sex of the chick nor to whether the 14C was in C-I or C-2 position. EI-14C]Glucose was metabolized to 14C02 at a faster rate than EI-14C] - galactose which differs from observations in the rat where the ~4COz curves are essen- tially the same. The intact chick was not able to convert [i-~4C]galactonate or [i-14Clgalactitol to 14COy Of the various tissues which metabolize [i-14C]galactose to 14C02 kidney converted the greatest amount.

INTRODUCTION

Numerous reports have appeared recently which describe various biochemical events associated with the neurotoxicity syndrome observed when the sugar D-galac- rose is fed to chicks. Galactitol I and galactose-I-phosphate ~ accumulate in brain and there is an alteration of brain phospholipid and adenine nucleotide metabolism 3. Enzymes of the uridine nucleotide pathway of galactose metabolism have been shown to be present in chick liver z, 4 and brain despite the fact that the chick is not normally exposed to dietary galactose.

The present work was undertaken to assess the functional operation of the galactose metabolizing enzymes in the chick by quantitation of the yield of 14C02 after administration of radioactive galactose to the intact animal. The oxidation of galactose has been compared to that of glucose and an evaluation of the oxidation of galactose metabolites, galactitol and galactonate, made. In addition, the conversion of [I-laC~galactose to 14C02 by various chick tissue in vitro has been examined. The results of these studies are contained in this report.

MATERIALS AND METHODS

Newly hatched Rhode Island Red chicks were obtained from Hall Brothers Hatcher, Watlingford, Conn., and maintained on non-medicated chick mash and wa- ter until utilized.

Biochim. Biophys. Acta, 222 (197 o) 327-332

328 H.J . WELLS et al.

[i-14CJGalactose (specific activity, IO mC/mM), I2-1aC~galactose (specific activity i . i mC/mM) and II-l*C~galactitol (specific activity, 45 mC/mM) were obtained from Calbiochem. Ii-14CJGalactonate (specific activity, o.i mC/mM) was synthesized by the procedure outlined by MOORE and LINK s. Glucose and Galactose were obtained from Pfanstiehl, St. Louis, Mo.

Experiments in vivo Solutions (o.i ml) of 5o mg of the sugar, containing o.5/~C of labeled sugar, were

injected intraperitoneally. The chick was then placed into an all glass metabolic chamber. The air leaving the chamber was passed over a cold finger containing a dry- ice-acetone mixture trapping out all water. Metabolic laCO2 was trapped by bubbling through a column containing I0 ml of hyamine-methanol ( I : I ; v/v). At periodic intervals, Io-minute collections were made, the column was drained, rinsed with small amounts of methanol, and the volume of hyamine-methanol made up to IO ml with the methanol rinse. Two-ml aliquots were added to 14 ml of scintillation fluid (Liqui- fluor, New England Nuclear Corp.) and counted in a Packard scintillation counter at 75% efficiency.

The amounts of x4CO, excreted in expired air by the chick were analyzed by the method of FREDRICKSON AND ONO e as modified by SEGAL et al 7.

Radioactivity of ~4CO2 was plotted as counts/min per min as a function of time. The cumulative 14CO2 was calculated by integration of the area under the CO 2 ex- cretion curve.

Experiments in vitro A Stadie-Riggs microtome was employed to make liver and kidney slices.

Because of the difficulty encountered in the removal of the friable chick kidney, use of only cortex slices was not at tempted. Small segments of everted jejunum were utilized. Brain slices were prepared with a MCILWAIN hand microtome s. About ioo mg of tissue were used in each incubation flask.

Oxidation experiments for collection and measurement of evolved ~4CO2 were carried out in modified Warburg flasks as described by WEINSTEIN and SEGAL 9. Tissues were incubated in 2.0 ml Krebs-Ringer bicarbonate buffer (pH 7.4) with i . i /,mole galactose containing 0.25/zC EI-liCJgalactose. 14CO~ was trapped in hyamine and assayed by a liquid scintillation technique as noted above.

RESULTS

IiCO, excretion after administration of radioactive galactose The 14CO 2 excretion of male and female chicks given tracer amounts of [1-14C~ -

galactose and [2-14C]galactose was independent of sex and position of the label given. Fig. IA illustrates a composite curve of the I4CO, excretion pat tern obtained after the administration of labeled galactose to male and female chicks. Other than varia- tions in peak height, this curve typifies the 1aco2 excretion curves obtained from chicks varying in age from I to 16 days. 14CO2 production rises gradually for 140 minutes, followed by a slow decline. After 5 hours there is still label detected as *aco2. Table I lists the cumulative 14C02 excretion of chicks ranging from I tot 16 days post-hatching.

Biochim. Biophys. Acta, 222 (197 o) 327-332

OXIDATION OF GALACTOSE IN THE CHICK 3 2 9

14C0 2 excretion after administration of ~I-14C]glucose In order to gain some insight as to the fate of the galactose label when it enters

the glucose metabolic scheme via the Leloir Pathway, II-14C]glucose was administered to male and female chicks. No differences in C02 excretion were noted between sexes. A composite curve of 14C02 production from glucose by male and female chicks is shown in Fig. IA.

Comparison of this curve with that obtained after the administration of galac- tose shows marked d~erences (Fig. IA). The 14C0~ excretion is very rapid, reaching

A 3000

1000 .E E

i i *

looo ~ . ~ _ . _ . ~ . _

5oc ~ - - ~ = ......

, , i i i i

5O 100 150 20O 250 300 Ti me (minutes)

Fig. IA. Composite of 14CO2 excretion of chicks, 7 days post-hatching, after the administrat ion of r ad ioac t i ve ga lac tose a nd glucose. 14CO 2 was col lected and t he curve was p l o t t e d as descr ibed in MATERIALS AND METHODS. E a c h po in t is the m e a n of 9 de t e rmi na t i ons . B. I4CO2 excre t ion curve o b t a i n e d a f te r a d m i n i s t r a t i o n of l abe led ga lac tose a nd glucose to the 4-day-old ra t . E a c h po in t is t he m e a n of 2 de te rmina t ions .

T A B L E I

E X T E N T OF O X I D A T I O N OF [ I - 1 4 C I G A L A C T O S E A N D [ I - 1 4 C ] G L U C O S E B Y I N T A C T C H I C K A N D R A T

Animal s were in jec ted i n t r a p e r i t o n e a l l y w i t h o . i m l of a so lu t ion con t a in ing 5 ° m g of the sugar p lus 0. 5 /zC of t r ace r t h e n p laced in a me tabo l i c chamber , x4co l was measu red as descr ibed in MATERIALS AND METHODS. Values r epor t ed represen t 5-h c u m u l a t i v e I4CO2 excre t ion and are expressed as a m e a n 4- s t a n d a r d error.

Age Average Number (days) wl. (g) of animals

% ofx4c administered expired as 14C02

Galactose Glucose

Chick i 36.5 4 25.8 ± 0.3 37.2 -t- 2.9 5 41.8 5 29.3 4- 1.3 51.3 4- 4.0 7 47 .0 9 27.2 4- o-3 56.8 4- 4.6 9 58.2 5 20.3 4- 0.9 35.4 4- 2.5

12 81.7 7 23.4 4- 0.7 37.9 • 3 .1 16 86. 7 6 23.6 4- 1.6 35.6 4- 2. 9

R a t 4 7.2 2 i o . i * lO.6 *

32 i2o 4 38.o 4- 3.o - -

* Values are the mean of 2 d e t e r m i n a t i o n s

Biochim. Biophys. Acta, 222 (197o) 327-332

330 H. j . WELLS et al.

a peak at 5 ° minutes then declining steeply. Production of ' 4 C O 2 w a s highest in the 5-7 day age group (Table I). Animals 9 days and older excreted less of the label as *aCO2. A similar pat tern of age-dependent production of CO 2 was not observed after the administration of labeled galactose.

Oxidation of EI-14Clgalactonate and [I J~Clgalactitol by the chick In order to determine the existence of an oxidative pathway for galactose meta-

bolism in the chick, [I-*4CJgalactonate, the metabolite of galactose via the oxidative pa thway *°, was administered. No label was detected in '4C02 during the 5-h experi- ment. Similar results were obtained after administration of [I-14Clgalactitol. No label was excreted as '4CO2, thus supporting the idea that galactitol is essentially a "dead- end" pathway for galactose metabolism 2.

Oxidation of EI-14Clgalactose and EI-l*C~glucose in the young rat EI-*4CJgalactose was administered to 4-day old rats in order that the ability

of the chick to metabolize galactose to CO 2 could be compared to an animal normally ingesting galactose. The '4C02 excretion curve obtained after the administration of [i-*4Qgalactose and [I-l*CJglucose to the neonatal rat is shown in Fig. IB. Compari- son of this curve with that from the chick shows some differences. The rat produces '4CO 2 rapidly, reaching a peak at 7 ° rain after injection of label, then declining to the end of the experiment. The chick, on the other hand, maintains its level of 14C02 excretion from 60 to 16o minutes after administration of label. Calculation of cumulative '4C02 showed the rat (6 g) excreting less 14C02 than the 36.5 g chick, lO.1% vs. 25.8%, respectively. If corrections for the differences in body weight or surface area were made, galactose metabolism in the rat relative to chick would be greater.

The administration of [I-*4Clglucose to the the rat resulted in a 14C02 excretion pat tern markedly different from that of the chick (Fig. IB). No peak occurred at 5 ° minutes as was observed with the chick. The curve paralleled that seen after the in- jection of galactose.

TABLE n OXIDATION OF GALACTOSE BY" CHICK TISSUE SLICES

T i s s u e s l ices w e r e i n c u b a t e d in 2.0 m l K r e b s - R i n g e r b i c a r b o n a t e buf fe r ( p H 7,4) c o n t a i n i n g I . I /2inoie a n d 0.25 /2C Ei-14C]galactose for 60 m i n a t 37 °. B r a i n t i s s u e w a s p r e i n c u b a t e d for 3 ° m i n a t 370 w i t h I i /*moles g lucose p r i o r t o t h e a d d i t i o n of g a l a c t o s e . 14CO~ w a s m e a s u r e d as d e s c r i b e d in MATERIALS AND METHODS. V a l u e s r e p o r t e d r e p r e s e n t a m e a n of 4 t r i a l s ~ s t a n d a r d error .

Tissue Sex Evolved 14C0 2 (nmoles per zoo mg wet weight)

B r a i n M 9.4 -ix 2.1 I ? 5.1 ± 1 - 9

L i v e r N[ 15.9 ! 4 .6 F 16.4 ~ 0-7

K i d n e y M 61.5 ± 11. 4 F 60.8 ~ 7.6

I n t e s t i n e Y[ 6. i * F 3.4 *

* V a l u e s a re t h e m e a n of 2 d e t e r m i n a t i o n s

Biochim. Biophys. Acta, 222 (197 o) 327-332

OXIDATION OF GALACTOSE IN THE CHICK 331

Oxidation of EI-l~Cigalactose by various tissues in vitro The rate of oxidation of EI-14Clgalactose by slices of brain, liver and kidney as

well as intestinal segments was assessed in order to obtain some insight into the rela- tive contribution of these tissues to the in vivo yield of 14C02 described previously. The results in Table I I reveal that the kidney possesses the greatest oxidizing capa- bility of these tissues. No significant difference in formation of 14CO2 was observed between tissues from males and females.

DISCUSSION

The demonstration of oxidation of [I-laClgalactose in intact chicks and by chick tissues in vitro together with the verified existence of galactokinase, uridyl- transferase and UDPGal-4-epimerase in chick tissues substantiates the capability of the chick to metabolize galactose despite the fact that this sugar is not normally a dietary consituent. Since EI-laClgalactonate and [I-laClgalactitol when given to the chick do not result in evolution of 14CO2 and there is no difference in 14CO2 excretion patterns after giving galactose labeled in C-I or C-2, it may be reasonable to conclude that the chick, like man, metabolizes galactose primarily via the sugar nucelotide pathway H.

Since the ultimate result of the latter pathway is conversion of galactose to glucose, the oxidation of both sugars was compared. In intact man, the curves of liberation 14C02 from [I-14Clgalactose and EI-14Clglucose are essentially equivalent 11. The present data indicate that the rat shares this same metabolic pattern. This does not appear to be the case for the chick which oxidizes glucose much more rapidly than galactose. Therefore, although chick tissues contain the sugar nucleotide pathway, its kinetic characteristics would appear to differ from that of the mammals examined being slow relative to glucose entry into its metabolic pathway. The slow entry of galactose into the glucose metabolizing system is underscored by the data of Table I which shows that there is a marked increase in ~aco 2 yield from glucose in the 5-7- day-old chick with no concomitant rise in galactose oxidation. The activity of enzymes of the galactose pathway in chick liver is less than that of the rat which is consistent with this interpretation. The cumulative excretion of 14CO2 from administered glucose observed in the 9-day and older chick agrees with the values of 26.2% and 34.2 % published by ALLRED AND KRATZER 12 and V~AGH AND WARBEL 13, respectively.

The analysis of [I-14C]galactose oxidation by tissues in vitro led to the surprising observation that the kidney most actively performed this conversion. Since kidney metabolism was 4-fold higher per unit weight and the total kidney weight is high relative to total liver weight 14, it would seem that the kidneys play a greater role in galactose metabolism than we had suspected. The relative importance of these tissues in mammals has not been evaluted.

In neither our in vivo nor in vitro oxidation studies have we been able to show a difference in galactose oxidation which is sex dependent to possibly account for the difference in the toxicity syndrome when galactose is fed 1. I t may be that this could be shown with use of larger amounts injected into the chick or used in the in vitro experiments. The lack of a sex difference in 14CO 2 liberation, despite the observed uridyltransferase difference in the livers of Rhode Island Red males and females,

Biochim. Biophys..4eta, 222 (197 o) 327-332

332 H.J . WELLS et al.

is consistent with the fact that galactokinase which shows no dependence on sex is the rate limiting enzyme 4.

Galactose toxicity produced in the chick appears to result not from a lack of metabolism of galactose but from overwhelming the normal capacity of the tissues to metabolize the large quantities of sugar administered. Whether the primary events in the new toxicity observed is the result of galactitol formation 1, galactose-i-phos- phate accumulation 2, 3, ATP depletion 3 or phospholipid abnormalities in brain re- mains to be determined.

ACKNOWLEDGEMENT

This work was supported by grants from the U.S. Public Health Service, AM 10894, and the Nutrition Foundation.

R E F E R E N C E S

i H. J. WELLS AND S. SEGAL, F E B S Letters, 5 (1969) 121. 2 J. S. MAYES, L. 1:{. MILLER AND F. K. MYERS, Biochem. Biophys. Res. Commun., 39 (197 o) 661. 3 L. P. KOZAK AND W. W. WELLS, Arch. Biochem. Biophys., 135 (1969) 371. 4 M. GORDON, H. J. WELLS AND S. SEGAL, submitted for publication. 5 S. MOORE AND K. P. LINK, J. Biol. Chem., 133 (194 o) 293. 6 D. S. FREDRICKSON AND K. ONO, jr. Lab. Ctin. Med., 51 (1958) 147. 7 S. SEPAL, A. BLAIR AND H. ROTH, Am. J. Med., 38 (1965) 62. 8 I-I. MclLWAIN AND R. RODNIGHT, in, Practical Neuroehemistry, Little, Brown & Co. (I969), p.

117 . 9 A. N. WEINSTEIN AND S. SEGAL, Biochim. Biophys. Aeta, 156 (1968) 9-

io P. CUATRECASAS AND S. SEGAL, Science, 153 (1966) 549. I i S. SEGAL AND P. CUATRECASAS, Am. J. Med,, 44 (1968) 34 o. 12 J. B. ALLRED AND F. H. KRATZER, Proc. Soc. Exptl. Biol. Med., 129 (1968) 658. 13 P. V. WAGH AND P. E. WARBEL, jr. Nutr., 92 (1967) 491. 14 W. R. BRENEMAN, Endocrinology, 28 (1941) 946.

Biochim. Biophys. Acta, 222 (197 o) 327-332