Glucitol pathway in human pregnancy

EUROP. J. OBSTET. GYNEC. REPROD. BIOL., 1978,8/6,323-328 0 Elsevier/North-Holland Biomedical Press

Glucitol pathway in human pregnancy P. Scirpa I, D. Mango l, A. Bompiani l and E. Menini ’ 1 Department of Obstetricsand Gynecology, and 2 Department of Biological Chemistry, Catholic University,Largo A. Gemelli 8, 00168 Rome, Italy

-SCIRPA, P., MANGO, D., BOMPIANI, A. and MENINI, E. (1978): Glucitol pathway in human pregnancy. Europ. J. Obstet. Gynec. reprod. Biol., 816, 323-328 The glucitol pathway in human pregnancy has been investigated by in vitro and in vivo studies. It has been shown in vitro that (a) the human placenta can transform glucose into glucitol, and to a lesser degree glucitol into fructose, and (b) the human fetal liver is capable of oxidizing glucitol into fructose. Furthermore, it has been found that glucitol concentration is higher in fetal than in maternal plasma at birth and that a significant arteriovenous difference (P < 0.05) exists in umbilical cord blood. The results of glucitol estimations in 40 specimens of amniotic fluid show a typical trend, with concentrations ranging between 400 and 900 pg/lOO ml from the 23rd to the 37th wk of gestation and a marked drop near term. These results indicate that in the human, as in the sheep, fructose can be formed in a ‘two stages in two organs’ process. The possible role of the glucitol pathway in human pregnancy is discussed. glucose; fructose; placenta; fetal liver; amniotic fluid; umbilical cord

Introduction

pregnancy, as also occurs to a much larger extent in the ungulates, there is a mechanism for net fructose production that resides in the placenta or in the fetoplacental unit. In mammalian tissues two pathways for the conversion of glucose to fructose have been demonstrated :

The fact that at birth human umbilical fetal blood contains more fructose than peripheral maternal blood (Hagerman and Wee, 1952), and the demonstration that human placental tissue in vitro can transform glucose into fructose (Hagerman and Villee, 1952; Hagerman, Roux and Villee, 1959; Ritter and Leuthardt, 1963), suggest that during human

Phosphohexose Glucose

w

Glucose z

G-6-P ,$?%?%s%

Glucitol

Alkaline F-6-P phosphata? Fructose

G~lu~de~tr~r

Fructose

323

(1)

(2)

P. Scirpa et al.: Glucitol in pregnancy

324

The first pathway involves the intermediates glucose6-phosphate and fructose-6.phosphate (Hagerman et al., 1959; Ritter and Leuthardt, 1963), and the second implies the reduction of glucose to D-glucitol (sorbitol) by the enzyme aldose reductase (alditol : NADP 1-oxidoreductase; EC 1.1 .1.21) followed by the oxidation of the hexitol to fructose by the enzyme glucitol dehydrogenase (L-iditol : NAD 5-oxidoreductase; EC 1.1.1.14) (Hers, 1956, 1957a; Samuels, Harding and Mann, 1962; Ritter and Leuthardt, 1963; Ellis, Strickland and Eccleston, 1973). The pathway involving phosphorylated intermediates is operative in all glycolytic tissues including the human placenta (Hagerman et al., 1959; Ritter and Leuthardt, 1963). Hers (1975a, b) has demonstrated that in the pregnant sheep the production of fructose, which is the most important source of energy for the fetus, takes place mainly through the glucitol pathway and that the first reaction of this process, the reduction of glucose to glucitol, occurs in the placenta while the second, the oxidation of glucitol to fructose, occurs in the fetal liver. As far as human pregnancy is concerned, the presence of Hers’ glucitol pathway - ‘two stages in two organs’ - has not as yet been demonstrated, although Ritter and Leuthardt (1963) and Mango, Scirpa and Menini (1976) have shown that the human placenta can form glucitol in vitro. The glucitol pathway is also operative in other mammalian tissues such as the seminal vesicles (Samuels et al., 1962), the large arterial wall (Clements, Morrison and Winegrad, 1969), the umbilical cord (Brachet, 1973), the lens (Van Heyningen, 1959) and the nervous tissue (Gabbay and O’Sullivan, 1966). At present, the quantitative significance of these two routes of&uctose formation in the human placenta or in the fetoplacental unjt has not been established, nor is the biological role of fructose or glucitol in human pregnancy known. This report presents the results of our studies on (a) the formation of fructose by subcellular fractions of human placenta by the glucitol pathway and by the glucosed-phosphate and fructose-6.phosphate pathway, (b) the in vitro transformation of glucitol to fructose by midpregnancy human fetal liver, and (c) the concentration of glucitol in arterious and venous umbilical cord blood, venous maternal blood

obtained at the time of delivery, and in amniotic fluid in normal pregnancies at different stages.

Material and methods

General reagents were of analytical grade and were purchased from Carlo Erba (Milan, Italy), BDH Chemicals Ltd. (Poole, Dorset, U.K.), Boehringer (Mannheim, F.R.G.), Merck (Darmstadt, F.R.G.) and Serva (Heidelberg, F.R.G.). Glucitol dehydrogenase, hexokinase (EC 2.7.1 .l), glucose-6.phosphate dehydrogenase (EC 1.l .1.49) and phosphoglucoisomerase (EC 5.3.19) were obtained from Boehringer (Manriheim, F.R.G.). Placental subcellular fractions (24,000 g supernatant, containing the mictosomal plus the soluble fraction) for in vitro studies were prepared from normal placentas, obtained immediately after vaginal delivery at term as described earlier (Mango et al., 1976). The fetal liver preparation was obtained from a 22. wk-old normal fetus which died 30 min after birth. A portion (2.2 g) of the liver was homogenized with 2 ~01s. of 0.155 M KC1 and centrifuged at 800 g. Before incubation the supernatant was extensively dialysed against 0.155 M KC1 at 4’C to remove low molecular weight substances. The subcellular suspensions of placenta and fetal liver were incubated with the appropriate substrates (see Results) at 37°C in a Dubnoff metabolic shaker, in Tris-HCl buffer, pH 7.4, with air as the gas phase. incubations in a total volume of 4.0 ml were allowed to proceed for 180 min. At the end of the incubation period, 5% ZnSO,, (2.0 ml) and 0.3 N Ba(OH)z (2.0 ml) were added to each flask and the resulting mixtures were centrifuged at 18OOg for 10 min. The supernatant was deionized by passage through an Amberlite IR-120 and IR4B column. The eluates were taken to dryness under vacuum in a desiccator. The recovery of this experimental procedure was 82.0% for glucitol and 94.9% for fructose respectively .

40 amniotic fluid samples were obtained by transabdominal amniocentesis or by puncture of the amniotic sac under visual control through the amnioscope, between the 23rd and the 41st wk of gestation. After amniocentesis the amniotic fluid was

P. Scirpa et al.: Glucitol in pregnancy

325

centrifuged at 1000 g for 20 min and the supernatant was kept at -20°C. Samples of fetal umbilical arterious and venous plasma and maternal venous plasma from normal pregnancies at term were also obtained at the time of vaginal delivery and the separated plasma was kept at -2O’C. Glucose, fructose, glucose-6-phosphate and fructose&phosphate were determined by enzymatic methods (Klotzsch and Bergmeyer, 1965). Glucitol was determined enzymatically according to WilliamsAshman (1965). All determinations were performed in duplicate. The specificity of the enzymatic determination of glucitol was controlled in a previous experiment by gas-liquid chromatography (GLC) (Mango et al., 1976). To obtain the complete enzymatic oxidation of glvcitol, the range of glucitol concentration in the deproteinized and deionized samples varied between 0.02 and 0.2 pmol/ml.

a series of incubations of placental preparations, corresponding to 0.8 g of wet tissue, with the following substrates and cofactors: (a) glucose and NADPH (generated from NADP, 5 ~01, and glucose-6phosphate, 20 ~01) and NAD; (b) glucitol and NADP and NAD; (c) glucose and ATF’;(d) glucose-6phosphate; (e) fructose-6-phosphate. At the end of the incubation the contents of the flask were analysed for the following products: (a) glucitol; (b) fructose; (c) fructose-6-phosphate, glucose-6phosphate and fructose; (d) glucose, fructosedphosphate and fructose; (e) glucose, glucose-6phosphate and fructose. The results of the two experiments (A and B) are shown in Table I. Analysis of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose and glucitol in the dialysed placental preparations used for the incubations showed only traces of glucitol (
In one experiment two aliquots of fetal liver preparation equivalent to 1 g of fresh tissue were incubated with glucitol (28 pmol) and NAD (5 ~01) as described in Material and Methods. At the end of the incubation period the medium was analysed for fructose. In the two incubations 1172 nmol and 608 nmol of this sugar were found, corresponding to a conversion of 43% and 2.2% of the initial amount of glucitol. Fructose could not be detected

Results

In vitro studies Placenta

Two identical experiments were carried out by incubating subcellular fractions of two normal placentas under the conditions described in Material and Methods. Each experiment consisted of

800

.

I

. 24

28

28

30 weeks

32 of

34

36

38

40

pregnancy

Fig. 1. Glucitol concentration in amniotic fluid at different stages of gestation.

, 42

P. Scirpa et al.: Glucitol in pregnancy

326 TABLE I

Production of glucitol, fructose, fructose&phosphate, glucose&phosphate plus soluble fractions, in two different experiments (A and B) Coenzymes

Substrates

and glucose by placental microsomal

nmol/g w.t.

Products

% conversion

A

B

A

B

(a) Glucose (28 rmol)

NADPH ’ NAD (5 rmol)

Glucitol

120

191

0.5

0.7

(b) Glucitol(28

pmol)

NADP (5 pmol) NAD (5 pmol)

Fructose

96

29

0.4

0.1

(c) Glucose (28

pmol)

ATP (12 pmol) MgClz (10 Mmol)

F-6-P G-6-P Fructose

29 n.d. n.d.

n.d. 18 n.d.

0.1 -

(d) G-6-P (30 pmol)

Glucose Fructose F-6-P

3549 868 n.d.

650 96 n.d.

11.8 2.8 -

2.1 0.3

(e) F-6-P (30 pmol)

Glucose Fructose G-6-P

3270 675 n.d.

361 48 n.d.

10.9 2.2 _

1.2 0.2 -

_ 0.06 -

n.d. = not detectable. 1 Generated from NADP (5 pmol) and G-6-P (20 I.cmol).

in the dialysed liver preparation used for the experiment .

arteriovenous difference exists in the cord blood, with glucitol concentrations in venous blood higher than in arterial blood. Figure 1 shows the results obtained after analysis of glucitol in samples of amniotic fluid at different stages of gestation. It can be seen that the concentration of glucitol remains between 400 and 900 E.cg/ 100 ml until the 37th wk and that in all cases it drops markedly near the term of gestation to values below 300 c(g/lOOml.

In vivo studies

Table II shows the concentration of glucitol in samples of venous and arterial cord blood and peripheral maternal blood. It is evident that the concentration of glucitol in the fetal plasma is higher than in the maternal plasma and that a significant

TABLE II

Umbilical cord and maternal plasma glucitol levels at vaginal delivery (@g/100 ml) Case:

UVP UAP MVP UVP-UAP difference UVP-MVP difference uvp = umbilical venous

1

2

3

4

5

6

7

8

9

M

SD

87.7 61.4 43.8 26.3 43.9

245.7 210.6 122.8 35.1 122.9

403.7 131.6 175.5 272.1 228.2

351.1 175.5 61.4 175.6 289.7

175.5 52.6 nd. 122.9 175.5

122.8 61.4 n.d. 61.4 122.8

118.5 105.3 105.3 13.2 13.2

193.1 67.5 n.d. 125.6 193.1

421.3 175.5 105.3 245.8 316.0

235.4 115.7 68.2 119.7 167.2

k127.4 259.9 k63.0 Pc0.05 P< 0.01

plasma;UAP= umbilical arterial plasma; MVP = maternal venous plasma; n.d. = not detectable.

P. Scirpa et al.: Glucitol in pregnancy

Discussion

The results of our in vitro experiments confirm that the human placenta at term is capable of transforming small amounts of glucose into glucitol, and to a lesser degree glucitol into fructose. The rate of ghrcose conversion into glucitol is similar to that previously found by us using the 14C substrate and GLC (Mango et al., 1976) in a series of human placentas. Moreover it may be seen that the human placenta in vitro can also produce fructose from glucose through glucose-6-phosphate and fructosedphosphate. These findings are similar to those of Ritter and Leuthardt (1963) but at variance with those of Hagerman et al. (1959) who could not demonstrate the transformation of glucose into glucitol. The higher content of glucitol in fetal plasma at birth with respect to maternal plasma and the existence of a significant (P< 0.05) arteriovenous difference in the cord blood as far as the concentration of this hexitol is concerned, are compatible with the concept that in vivo, as it occurs in vitro, the production of glucitol in the human placenta is higher than the average production of this compound in the mother and that placental glucitol is used up by the fetus. These results, taken together with the demonstration in vitro that human fetal liver is capable of oxidizing glucitol to fructose, suggest that, in a similar manner as in the pregnant sheep (Hers, 1957a,b), in the pregnant woman fructose can also be formed by a two-stage process in two different organs, the placenta and the fetal liver. The presence of glucitol in amniotic fluid, taken as a fetal compartment, at various stages of gestation, together with the fact that fetal plasma contains more glucitol than maternal plasma, suggest that this substance may play some role in the human fetal metabolism, which we feel deserves further attention. The concentration of glucitol in amniotic fluid at different stages of pregnancy shows a typical trend, similar to that found for glucose (DraZanEik and KuvaEiC, 1974) in the same compartment. Glucitol concentrations vary between 400 and 900 pg/lOO ml from the 23rd to the 37th wk, then drop markedly to less than 300 E.cg/lOOml near term. The latter value is similar to the concentration of glucitol found

327

in fetal plasma at birth. The decrease in the concentration of glucitol in amniotic fluid near term could be explained in terms of decreased placental production or increased fetal utilization as a consequence of increased activity of the fetal liver enzymes (Pikkarainen and Raiha, 1967). As far as the role of glucitol in human pregnancy is concerned, it could be that, as occurs in other animal species (Horecker, 1968), the glucitol pathway represents for the human fetus an alternative route to the metabolism of glucose. It is likely that in the normal pregnant woman this route of glucose utilization is of relatively minor importance, but it could become more important in conditions associated with impaired glucose utilization. Another possible role of glucitol could be the regulation of NADP/NADPH equilibrium, as has been proposed by Brachet (1973) in the umbilical cord tissue. However, the microgram quantities of glucitol recovered in the biological fluids, as well as the rate of placental production, suggest that the glucitol pathway in human pregnancy may only represent the persistence of a vestigial reaction, which can be studied for diagnostic purposes.

References Brachet, E.A. (1973): Presence of the complete sorbitol pathway in the human normal umbilical cord tissue. Biol. Neonat. (Basel), 23. 314.

Clements, R.S., Morrison, A.D. and Winegrad, A.I. (1969): Polyol pathway in aorta. Regulation by hormones. Science,

166, 1007.

Draian%, A. and Kuva%, I. (1974): Amniotic fluid glucose concentration. Amer. J. Obstet. Gynec., 120, 40. Ellis, D.A., Strickland, J.M. and Eccleston, J.F. (1973): The direct interconversion of glucose and fructose in human skeletal muscle with special reference to childhood muscular dystrophy. CIin. Sci., 44, 321. Gabbay, K.H. and O’Sullivan, J.B. (1966): Sorbitol pathway: presence in nerve and cord with substrate accumulation in diabetes. Science, 1.51, 209. Hagerman, D.D. and Villee, C.A. (1952): The transport of fructose by human placenta. J. c&n. Invest., 31, 911. Hagerman, D.D., Roux, J. and Villee, C.A. (1959): Studies of the mechanism of fructose production by human placenta. J. Physiol. (Lond.), 146, 98. Hers, H.G. (1956): Le me’canisme de la transformation de glucose en fructose par les visicules seminales. Biochim. biophys. Acta, 22, 202. Le mttabolisme

Hers, H.G. (1957a): Bruxelles.

du fructose.

Arscia,

328 Hers, H.G. (1957b): Presence of sorbitol in seminal vesicles and foetal blood of the sheep. Biochem. J., 66, 30P. Horecker, B.L. (1968): Two major pathways and the interconversion of sugars. In: Carbohydrate Metabolism and its Disorders, Vol. 1, p. 158. Editors: F. Dickens, P.J. Randle and W.J. Whelan. Academic Press, LondonNew York. Klotzsch, H. and Bergmeyer, H.U. (1965): In: Methods of Enzymatic Analysis, p. 156. Editor: H.U. Bergmeyer. Academic Press, New York-London. Mango, D., S&pa, P. and Menini, E. (1976): Effects of dehydroepiandrosterone 16a-hydroxydehydroand epiandrosterone on the reduction of glucose to glucitol by the human placenta. Horm. Metab. Res., 8, 302. Pikkarainen, P.H. and Raiha, N.C.R. (1967): Development of alcohol dehydrogenase activity in the human liver. Pediat. Res., 1, 165.

P. Scirpa et al.: Glucitol in pregnancy Ritter, P. and Leuthardt, F. (1963): Die Bildung von Fructose und Sorbit aus Glukose in der menschlichen Placenta. Helv. physiol. Acta, 21, 212. Samueis, L.T., Harding, B.W. and Mann, T. (1962): Aldose reductase and ketose reductase in male accessory organs of reproduction. Biochem. J., 84, 39. Van Heyningen, R. (1959): Formation of polyols by the lens of the rat with sugar cataract. Nature (Lond.), 184, 194. Williams-Ashman, H.G. (1965): In: Methods of Enzymatic Analysis, p. 167. Editor: H.U. Bergmeyer. Academic Press, New York-London. Reprint requests and correspondence to: Dr. Paolo Scirpa, Department of Obstetrics and Gynecology, Catholic University, Largo A. Gemelh 8,00168 Rome, Italy.