�9 1997 by Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/97/5801-2-0085 $9.50

Plasma Glutathione Peroxidase Activity and Selenium Levels of Newborns with Jaundice

AHMET AYDIN,* AHMET SAYAL, AND A~KIN I~IMER Department of Pharmaceutical Toxicology, Ghlhane Military

Medical Academy, 06010 Etlik Ankara, Turkey Received December 19, 1995; Revised May 5, 1996;

Accepted July 1, 1996

ABSTRACT

The plasma glutathione peroxidase (GSH-Px) and selenium (Se) levels were determined in 31 newborns affected by jaundice (N~). The GSH-Px levels of both full-term and premature newborns exhibiting jaundice and having a birthweight lower than 2000 g were significantly low (p < 0.05) when compared to controls. No significant differences were found in the corresponding Se levels, which were similar in all groups and independent of the pregnancy period and birthweight.

Index Entries: Selenium; glutathione peroxidase; newborn jaundice.

INTRODUCTION

Jaundice is a condition that occurs because of fragmentation of ery- throcytes leading to accumulation of excess bilirubin. This leads to yellow pigmentation of sclera, mucosa, and skin of newborns. There are several causes for erythrocyte rupture, one of which involves oxygen and hydroxyl radicals destroying of the erythrocyte wall. Other causes involve iron transport disorders, liver deficiency, or excretion disorders (1).

In newborns, this condition is particularly dangerous, since it may lead to brain injury if undetected for periods of 48 h or more. Physio- logical disorders leading to jaundice occur in the third to fifth days after birth, although jaundice from other causes, such as neonatal hepatitis, biliary atresia, pylorostenosis, and hypothyroidism, may occur at a latter stage and will usually last for longer periods of time.

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 8 5 Vol. 58, 1997

86 Aydin, Sayal, and l~imer

Against oxidative damage, cells rely on a system that involves glu- tathione peroxidase (GSH-Px), superoxide dismutase, catalase, glu- tathione reductase, selenium (Se), and vitamins A, C, and E (2,3). The enzyme GSH-Px is present in various amounts in the plasma, erythro- cytes, thrombocytes, placenta, and in other tissues (4), and helps to pro- tect the cell from reactive oxygen intermediates.

Se is a well-known essential trace element that serves as a catalytic center in GSH-Px, thus having an essential part in the antioxidative defense system. Se intake is directly related to GSH-Px activity and dis- tribution throughout the body (4).

Newborns are exposed to oxidative stress from oxygen radicals pro- duced during birth in the erythrocytes themselves. It has been suggested that in cases where there is a weak antioxidative system, hemolysis may lead to newborn jaundice (5,6), although there is some controversy around this subject (7,8).

To test the hypothesis that newborn jaundice (NWJ) arises from oxidative damage to erythrocytes, and because Se supplementation may be of help to the antioxidative defense system, we have studied whether Se levels and GSH-Px activity are indeed involved in this condition.

MATERIALS AND METHODS

Thirty-one newborns who were affected by jaundice by the fourth day after birth were selected for this study. Fourteen were full-term babies, and the remaining 17 were premature. The control group consisted of 12 normal healthy babies. All subjects were born and cared for at the Zekai Tahir Burak Gynecology and Obstetrics Hospital, Ankara, Turkey.

Birthweight, birth term, and bilirubin levels of all newborns were determined by standard laboratory techniques. The NWJ subjects were separated into four groups according to the ethiology of jaundice as fol- lows: group 1 (n = 8) consisted of newborns affected by Rh-factor incom- patibility; group 2 (n = 11) by ABO incompatibility; group 3 (n = 3) because of sepsis; and group 4 (n = 9) because of physiological jaundice.

The NWJ were also classified by weight: group A with weight lower than 2000 g (n = 10); group B, weighing between 2000 and 3000 g (n = 13), and group C, with weight over 3000 g (n = 8). Finally, three more groups were formed according to bilirubin levels: group B1 (<10 mg/dL, n = 8); group B2 (10-15 mg/dL, n = 16), and B3 (>15 mg/dL, n = 7).

Blood was drawn from the antecubital vein and collected in heparinized polypropylene tubes. The plasma was separated by cen- trifugation for 5 min at 4000g, and used for GSH-Px activity analysis by spectrometry, whereas Se was determined by atomic absorption by tech- niques previously reported (9,10).

The instruments utilized were a Philips PU 8630 spectrometer and a Varian 30/40 electrothermal atomic absorption spectrometer provided

Biological Trace Element Research Vol. 58, 1997

Plasma S e a n d GSH-Px Ac t i v i t y in N e w b o r n s

Table 1 Plasma GSH-Px and Se Levels of NWJ and Controls

(Mean Value _+ SD) a

87

NWJ Controls

GSH-Px Activity (U/ml) 0.365+0.079 0.429~0.084

Selenium (ng/ml) 36.82• 10.11 40.13• 15.63

aSignificance of difference with controls p < 0.05.

Table 2 Plasma GSH-Px Activity and Se Levels of NWS and Controls a

Ethiology GSH-Px Activity Se Levels

(U/ml) (ng/ml)

Kh Incompatibility 0.357• 27.26•

ABO Incompatibility 0.338• 40.34•

Sepsis 0.346+0.160 32.16~: 1.66

Physiological Jaundice 0.350• 40.29•

Controls 0.429a:0.084 40.13 + 15.63

aSignificance of difference with controls p < 0.05.

wi th a m o d e l GTA-96 graphi te tube atomizer. Pal ladium-ascorbic acid was used as a chemical modi f ie r (10).

Statistical evaluat ions were obta ined by the M a n n - W h i t n e y U-test.

RESULTS

The results are s u m m a r i z e d in Tables 1-5. In Table 1, it is seen that GSH-Px activity in NWJ is significantly lower than that of the controls, p < 0.05. The cor respond ing values are 0.36 + 0.079 and 0.429 _+ 0.084 U / m L , respectively. By contrast, no significant differences were found for the p lasma Se levels, 40.13 + 15.63 n g / m L .

Table 2 gives values for the p lasma GSH-Px activity and Se levels in newborns according to the jaundice ethiology. In all cases except for jaun-

Biological Trace Element Research Vol. 58, 1997

88 Ayd in , Sayal , a n d l~imer

Table 3 Plasma GSH-Px and Se Levels of NWJ: Premature vs Full-Term

Subjects (Mean Value + SD) a

GSH-Px Activity (U/ml)

Selenium (ng/ml)

Premature Full-Term

(n=14) (n=17)

0.355+0.075 0.395•

40.09a:8.78 40.34i7.75

aSignificance of difference with full-term newborns p < 0.05.

Table 4 Plasma GSH-Px and Se Levels of NWJ Classified by Birthweight

(Mean Value _+ SD) a

Weight at Birth

<2000 g 2000-3000 g >3000g

GSH-Px Activity 0.369-&0.008 0.3664-0.059 0.356•

Selenium (ng/ml) 40.8 t • 36.42-4:9.73 32.43+ 11.98

aNo significant differences were found.

dice from sepsis, GSH-Px activity values are significantly lower than con- trols, p < 0.05, whereas Se levels are significantly lower (p < 0.05) when Rh incompatibility is the cause of the disease.

The results in Table 3 are for the groups classified according to preg- nancy period. The GSH-Px activity of premature NWJ babies is lower than that of full-term subjects, p < 0.05. Se levels are not significantly dif- ferent in both groups.

Table 4 lists GSH-Px activities and Se levels for NWJ according to birthweight. No significant differences are found between these groups. Finally, Table 5 gives the relationship between plasma GSH-Px activity, plasma Se and bilirubin levels. For NWJ showing bilirubin levels above 15 mg/dL , both plasma Se and GSH-Px activity are significantly lower than the 10-15 m g / d L group, (p < 0.05). The observed value for the <10 m g / d L group is higher than that of the >15 m g / d L group, although not enough to be statistically significant.

Biological Trace Element Research Vol. 58, I997

Plasma Se a n d GSH-Px Act iv i ty in N e w b o r n s

Table 5 Plasma GfiH-Px, Se and Bilirubin Levels of NWJ (Mean Value + riD)

89

GSH-Px Activity (u/ml)

Selenium (ng/ml)

Bilirubin Range

<10 mg/dl 10-15 mg/dl >15 rng/dl (n=8) (n=16) Cn=7)

0,360a:0,083 0.378+0.077 0.350-~0.018"

38.47+9.22 39.04• 31.41•

*Level of significance p < 0.05 when compared to 10-15 mg/dL group.

DISCUSSION

It is known that the biochemical function of the enzyme GSH-Px is to reduce most organic and inorganic hydroperoxides in many tissues and cells (4). These hydroperoxides may be generated by the reaction of free radicals with molecules, such as DNA and lipids, among others. Newborns are subject to increased oxidative stress, and in cases where there is a depressed antioxidant system, i.e., lower GSH-Px activity and lower Se levels, the reactive oxygen intermediates formed in the ery- throcytes may attack their membrane, leading to jaundice (2,7).

Bracci et al. reported that GSH-Px and other antioxidative enzymes are significantly depressed in NWJ (5). They also showed a negative cor- relation between bilirubin levels and GSH-Px activity. Pati et al. recog- nized an increased incidence of jaundice, up by 5.4%, in subjects with normal reduced glutathione levels and up by 12.8% in subjects with GSH-Px enzyme deficiency. They suggested that GSH-Px deficiency may cause hyperbilirubinemia in newborns (6). Tubman et al. reported values of GSH-Px of 2.74 _+ 0.08 ua/mL and showed that it is unrelated to body weight and birth period (11). Our GSH-Px values did not corrolate with birthweight as Tubman's result.

Some authors have suggested that Se levels are neither related to birthweight nor pregnancy period. For example, Rudolph et al. found no significant differences in the Se levels of underweight newborns fed with various infant formulas. Since Se is known to be at the active site of the enzyme GSH-Px (13-15) and is involved in cell membrane protection, playing a protective role in many diseases (15-18), we expected signifi- cant differences in NWJ when compared to healthy subjects. These were not found, however, as described in the results.

Our results seem to confirm previous reports of depressed GSH-Px enzyme activity in NWJ. The only exception was when the disease was attributed to bacterial infection, which is in agreement with the sugges-

Biological Trace Element Research Vol. 58, 1997

90 Aydin, Sayal, and l~imer

tion that it arises from an external, rather than a physiological factor. Also, the fact that there is an inverse relationship between bilirubin lev- els and GSH-Px activity indicates that jaundice may be owing to red cell membrane rupture by reactive oxygen species.

REFERENCES

1. R. E. Behrman and V. C. Vaughan, in Nelson Textbook of Pediatrics, Igaku-Shoin/Saun- ders, Tokyo, p. 355 (1983).

2. L. Friedvich, Science 201, 805-880 (1978). 3. A. Miester and M. E. Anderson, Ann. Rev. Biochem. 52, 711-760 (1983). 4. G. Perona, R. Schiavon, G. C. Guidi, D. Veneri, and P. Minuz, Thrombosis and Hemo-

stasis, 64, 312-318 (1990). 5. R. Bracci, G. Buonocore, B. Talluri, and S. Berin, Acta Pediatrica Scand. 77, 349-356

(1988). 6. H. P. Pati, M. Singh, V. K. Paul, R. K. Gupta, and A. K. Saraya, J. Trop. Med. Hygiene

93, 290-294 (1990). 7. S. Fuji, Br. J. Haematol. 68, 263-271 (1988). 8. S. Fuji, G. L. Dale, and E. Beutler, Blood 63, 1096-1101 (1984). 9. P. A. Pleban, A. Munyani, and J. Beachum, J. Clin. Chem. 2, 311-316 (1982).

10. M. B. Knowles and K. G. Brodie, J. Anal. Atomic Spectrom. 3, 511-516 (1988). 11. T. R. Tubman, H. I. Halliday, and D. McMaster, Biol. Neonate 58, 305-310 (1990). 12. N. M. B. B. ch. Rudolph and O. M. D. Preis, J Pediatr. 99, 57-62 (1981). 13. W. Mertz, Science 213, 1332-1338 (1981). 14. T. C. Stadman, Ann. Rev. Biochem. 49, 93-110 (1980). 15. J. T. Salonen, Ann. Clin. Res. 18, 18-21 (1986). 16. M. E Robinson and C. D. Thomson, Nutr. Abs. Rev. Clin. Nutr. 53, 6--26 (1983). 17. G. Yang, S. Wang, R. Zhon, and S. Sun, Ann. J. Clin. Nutr. 37, 872-881 (1983). 18. E. Delibas, B. Turan, E. Yiicel, R. Sasmaz, A. Isimer, and A. Sayal, Biol. Trace Element

Res. 28, 21-25 (1991).

Biological Trace Element Research Vol. 58, 1997