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Journal of the American College of Nutrition, Vol. 19, No. 2, 237-241 (2000)
Published by the American College of Nutrition

Original Research

Effects of Early Nutrition on Free Radical Formation in VLBW Infants with Respiratory Distress

Erika Tomsits, MD, PhD, Katalin Rischák and Lajos Szollár

2nd Department of Pediatrics, Department of Pathophysiology, Semmelweis University of Medicine, Budapest, HUNGARY

Address reprint requests to: Erika Tomsits, MD, PhD, II. Department of Paediatrics Semmelweis University of Medicine, H-1094 Budapest, Tuzoltó u. 7-9., HUNGARY


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Objective: We studied the development of essential fatty acid deficiency (EFAD) and its effects together with those of vitamin E deficiency on the free radical formation of very low birth weight (VLBW) infants with respiratory distress.

Methods: Infants were divided into three groups based on the way each was supplied with daily total energy intake: (1) by fat free parenteral nutrition only or by nutrition composed of (2) less than or (3) higher than 25% of total daily energy intake given in oral feeding. We measured plasma lipid parameters and autoxidative susceptibility (AOS) of red blood cells (RBCs).

Results: Plasma concentrations of linoleic acid were low in all the groups. After at least 14 days of feeding, eicosatrienoic acid (EA) was not detected. One week after the introduction of oral feeding, the abnormal triene/tetraene ratio of the groups had decreased, but was not normalized. Vitamin E deficiency was associated with significantly increased AOS, but EFAD was not. The two factors together caused an increase of AOS, that was additive.

Conclusions: Our data confirm that EFAD increases AOS of RBCs in VLBW infants. We assume that prevention of EFAD in VLBW infants could decrease the prevalence of complications associated with free radical formation.

Key words: EFAD, nutrition, autoxidative susceptibility of RBCs, TBARS, vitamin E, VLBW infants


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro, the cytotoxic effect caused by free radical formation can be reduced by the administration of certain unsaturated fatty acids (UFA), while other unsaturated fatty acids result in the opposite effect [1,2]. Clinical studies have shown that the administration of lipid emulsions containing an excess of UFA to immature infants causes a higher frequency of disorders thought to be related to increased free radical formation (e.g., bronchopulmonary dysplasia and retrolental fibroplasia). The essential fatty acid deficiency—which is a type of deficiency of unsaturated fatty acids—may influence free radical formation in very low birth weight infants too. To assess free radical formation we measured the autooxidative susceptibility (AOS) of red blood cells (RBCs) in premature infants with respiratory therapy. We determined the concentration of thiobarbituric acid reacting malondialdehyde substances (TBARS) in RBCs, as an index of AOS. We also studied the effect of vitamin E deficiency, alone and with essential fatty acid deficiency (EFAD) on the AOS of the RBCs of very low birth weight (VLBW) infants.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Twenty-two VLBW infants treated at the Departments of Pediatrics and of Obstetrics and Gynecology of the Semmelweis University of Medicine were included in the study. The procedures were in accord with the ethical standards of the Hungarian National Ethical Committee on human experimentation. On days one, seven and 21 of life, together with the necessary blood drawing and the written consents of their parents, an extra 1 ml of blood was taken through the cannula of the umbilical artery or from a peripheral vein. All the premature infants exhibited normal intrauterine development. The birth weights of premature infants were 1,263±231 g, the gestational age 30.2±1.32 weeks. Six patients had infant respiratory distress syndrome (IRDS), ten infants had perinatal infection or pneumonia, four premature infants had wet lung, five infants cerebral hemorrhages. Two of the patients had IRDS and sepsis, and three of them IRDS and cerebral hemorrhages together. Nutrition and vitamin E supplementation of newborns were carried out by the physicians according to routine protocols of the clinics, independently of our study. Twelve infants were given 20 mg/kg/day vitamin E orally throughout the study.

We divided the children into three groups according to the nutrition of the infants. In the first group, newborns got only fat free parenteral nutrition (P), i.e., infusion of glucose, NaCl and amino acids. The second and third groups received oral plus parenteral nutrition. In the second group, the energy provided orally was less than 25% (L) and, in the third group, higher than 25% (H) of the daily energy intake. The oral feeding was by breast-milk. All infants needed positive pressure ventilation from the first day of life. The duration of the mechanical support was not significantly higher in the first group than in the other two groups, (14.2±3.4 days vs. 12.8±2.9 days). We included infants in the study if, throughout the study, arterial pO2 was less than 98 mmHg and capillary pO2 was more than 40 mmHg.

Blood was drawn three hours postprandially in those infants on oral feeding, adding 1 mg/mL EDTA, and was immediately analyzed. Lipids determination was done following the Folch extraction using a 1:1 mixture of chloroform and methanol [3]. We used phosphovanillic color reaction for the measurements of plasma lipid concentrations. Plasma total tocopherol level was determinated by Quaife’s method [4]. Vitamin E/total lipid ratio [5,6] was used to determine the vitamin E status in the VLBW infants. Vitamin E deficiency was evaluated if the plasma concentration was less than 0.69 mg/dL or vitamin E/total lipid ratio was less than 1.9. To examine the AOS of RBCs, malondialdehyde products reacting with thiobarbituric acid (TBARS), released from red blood cell membranes due to hydrogen peroxide provocation, were measured [7]. Plasma total lipid samples were stored at -20°C until the gas chromatography procedure, using 10% SP2340 Supelcoport 100/120 (1.7 mx2 mm) column (T inj: 250°C, T det: 250°C, T oven: progr 170–230°C, 3°C/min), flame ionizing detector and SP 4290 (Spectrophysics, San Jose, California) integrator. Fatty acids 20:3 and 20:4 were identified using standard samples from Supelco Inc. The plasma eicosatrienoic/arachidonic acid (EA/AA) ratio was plotted against age. We defined EFAD if the ratio was more than 0.2.

Statistical Analysis
All data are presented as means±SEM. Statistical evaluations were performed by using ANOVA in STATGRAPHICS 4.0 (STSC Inc); p values of less than 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma total lipid concentration significantly increased after the introduction of oral nutrition. It was not significantly influenced by the age throughout the experiment (P: day 1 (n=20); 248±49 mg/dL, day 7 (n=11); 297.8±54 mg/dL, p>0.05; L: day 1 (n=2); 286±57.8 mg/dL, day 7 (n=7); 328.6±61 mg/dL; P:L p<0.05 on day 7; H: day 7 (n=4); 332±48 mg/dL, P:H p<0.01).

The fatty acid composition of the plasma lipid fraction of one, seven and 21 day-old preterm infants is shown in Table 1. Linoleic acid (LA) concentrations measured in all of our samples were lower than those of premature infants studied by Farrell [8]. On day 21, we were unable to detect eicosatrienoic acid in only four children, who had already been receiving L nutrition since day seven.


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  		Table 1. Alteration of Plasma Fatty Acid Composition in Preterm Infants

 
In the majority of examined preterm infants with lipid free parenteral nutrition, laboratory parameters of EFAD were found to be developed already on day seven. On day one only one child among the premature infants exhibited a pathological EA/AA ratio greater than 0.2, on day seven 63% did (Fig. 1).



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  		Fig. 1. Effect of nutrition on plasma EA/AA ratio plotted against the age. By day 21, all the infants in P group exhibited pathological plasma EA/AA ratio and the majority of this group showed pathological EA/AA ratio already on day seven. Closed circles represent lipid free total parenteral nutrition, closed triangles represent H infants and open circles mark the L group. The dotted line represents the lower limit of EFAD.

 
The connection between age, vitamin E supply and plasma vitamin E concentration is shown in Fig. 2. With age, plasma vitamin E concentration was increased in both vitamin E supplemented and non-supplemented groups, but in the former group, we measured higher values on days seven and 21 (0.48±0.16 mg/dL vs. 0.78±0.31 mg/dL; p<0.01; 0.78±0.22 mg/dL vs. 1.16±0.38 mg/dL; p<0.01, respectively). The introduction of oral nutrition elevated plasma vitamin E/total lipid ratio in newborns with, as well as without, vitamin E supplementation (Fig. 3).



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  		Fig. 2. Alteration of plasma vitamin E concentration with aging. Significant difference could be observed between vitamin E supplemented and unsupplemented groups from day seven onwards. Closed circles represent vitamin E supplementation, open ones show nonsupplemented infants. The dotted line stands for the upper limit of vitamin E deficiency.

 


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  		Fig. 3. Connection between changes of plasma vitamin E/total lipid ratio and aging. Plasma vitamin E/total lipid ratio increased during the course of aging. The dotted line represents 1.9 of normal plasma vitamin E/total lipid ratio. Closed circles represent vitamin E supplementation, open ones show non supplemented infants. **p<0.01.

 
The amount of TBARS was 795±67 nmol malondialdehyde/g Hb in VLBW infants with neither essential fatty acid nor with vitamin E deficiency. This group was considered the control. Pathological EA/AA ratio alone did not cause a significant increase in AOS of RBCs (825±78 nmol MDA/g Hb). Vitamin E deficiency significantly elevated AOS of RBCs (1,045±98 nmol/g Hb, p<0.01). Essential fatty acid and vitamin E deficiency together further increased TBARS concentration (1,150±102 nmol/g Hb, p<0.005; Fig. 4).



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  		Fig. 4. Effects of the parameters characterizing vitamin E and essential fatty acid status of preterm infants on plasma TBARS concentrations of red blood cells. Pathological EA/AA ratio alone did not alter plasma TBARS concentration. Vitamin E deficiency significantly elevated plasma TBARS concentration compared to control (p<0.01). Infants with both vitamin E deficiency and with pathological EA/AA ratio exhibited plasma TBARS levels (compared to control p<0.005; compared to the vitamin E deficiency group p<0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Limited energy reserves as well as the increased energy requirements of VLBW infants necessitates a high level of energy intake at a very early age [9–11]. Enteral nutrition encounters difficulties, owing to the infants’ immaturity. Besides, prolonged respiratory therapy to cure their respiratory distress further delays the introduction of per os nutrition [12–14]. Fat free parenteral nutrition of these newborns may lead to an early onset of EFAD [8,15–18] and to the development of low energy intake [19] due to problems affecting their energy metabolism, i.e., low glucose tolerance [20], limits of fluid administration, inclination to N-retention [21–23]. The energy deficiency causes utilization of linoleic (18:2n-6) and linolenic (18:3n-3) acids [24]. Relative deficiency of linoleic and linolenic acids leads to lower rate of synthesis of arachidonic (20:4n-6) acid (AA) and docosahexaenoic (22:6n-3) acid (DHA), which are necessary membrane components of the cells [25], mostly in the central nervous system and in the retina [26,27]. They are also important for prostaglandin synthesis [28–31]. To confirm EFAD, the quantity of LA and eicosatrienoic (20:3n-9) acid, as well as the triene/tetraene ratio (EA/AA), from total plasma lipid [8] were determined. The latter procedure rather indicates essential fatty acid intake, and it characterizes essential fatty acid homeostasis only in starving individuals [24]. Abnormal EA/AA ratio shows EFAD primarily due to its low concentration in the food and/or impaired function of delta-6-desaturase. It is not a convenient marker in cases with low production of DHA and AA, owing to a late enzyme (e.g. elongation enzymes, delta-5-desaturase) malfunction [24]. To determine essential fatty acid concentration here, using the melting point method is much preferred [32,33].

Complications (e.g., bronchopulmonary dysplasia, retrolental fibroplasia), partly related to the release of free radicals, are quite frequently found in premature infants in need of respiratory therapy (hyperoxic condition) [34,35]. Also, EFAD may develop in VLBW premature infants.

In the present study, low plasma LA levels seem predictive of EFAD, even on day 21 in all the VLBW infants. The abnormal presence of eicosatrienoic acid could be detected in all premature infants except those who already received oral feeding from the tenth day onward. An EA/AA ratio of more than 0.2, characteristic of EFAD, was measured in 63% of the newborns on day seven, as well as in two thirds of the babies in the fat free nutrition group. There was no infant on the 21st day in the H group who had an EA/AA ratio more than 0.2, and the EA/AA ratio was similar to those infants in the L group who had been fed by breast milk at least the previous ten days. Significant differences can be observed between vitamin E supplemented and nonsupplemented groups. After seven days of fat free parenteral nutrition, 82% of infants in the vitamin E nonsupplemented group had lower vitamin E concentrations; vitamin E, whether expressed as total concentration or in a ratio with total serum lipids, was lower than normal.

On the 21st day the vitamin E concentration was significantly different in the vitamin E supplemented group than in the nonsupplemented. This time the infants in the vitamin E nonsupplemented group had vitamin E concentrations in the normal range. The vitamin E deficiency can increase the TBARS concentration significantly in the first week of life (Fig. 3).

Our result could be influenced by differing durations of mechanical ventilation; we tried to increase our sample size to exclude this possibly effect [40].

EFAD alone could not significantly increase the AOS, but the EFAD together with the vitamin E deficiency caused higher elevation than the vitamin E deficiency alone. Although vitamin E supplementation can not reduce stages III and IV bronchopulmonary dysplasia [41], the prevention of EFAD in VLBW infants could decrease the production of the free radicals.


   FOOTNOTES
 
This work was in part supported by grants ETK 7/451 of the Ministry of Health, OTKA 185 and OTKA 1057 of the National Scientific Research Foundation, and ETT 191 of the Ministry of Public Welfare.

Received October 1, 1998. Accepted October 1, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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This Article
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