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Altered Cord Serum Lipid Levels Associated With Small for Gestational Age Infants

From the Departments of Obstetrics and Gynecology and Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky.

Address reprint requests to: Douglas D. Taylor, PhD, Division of Gynecologic Oncology, University of Louisville School of Medicine, 511 South Floyd Street, MDR 420, Louisville, KY 40202, E-mail: ddtaylor{at}juno.com

	Abstract

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Objective: To determine whether small for gestational age (SGA) infants show changes in lipid metabolism that could distinguish growth-restricted subpopulations.

Methods: Sera from the arterial cord blood from 38 SGA infants were analyzed for apolipoprotein A-I level, total lipid content, and distribution of those lipids as triglycerides, diglycerides, free fatty acids, and phospholipids. Comparisons were made between appropriate for gestational age (AGA) controls (n = 25), SGA infants with a ponderal index below the tenth percentile (SGA I, n = 20), and SGA infants with a ponderal index above the tenth percentile (SGA II, n =18).

Results: Total cord serum lipid content was markedly decreased in all SGA infants compared with AGA infants (2.8 times lower). Although SGA infants showed total lipid concentration decreases, SGA I and SGA II infants showed distinct characteristics. Infants in the SGA I group had higher triglyceride levels (1.8 times higher) and lower free fatty acid levels (1.4 times lower), compared with AGA infants (P < .001). The lipid subclass distribution in SGA II infants was not significantly different from that in AGA infants, with the exception of an increase in triglyceride concentrations (1.3 times higher). Although the 22-kD placenta-derived apolipoprotein A-I was similar in all groups, the level of fetal liver–derived 28-kD apolipoprotein A-I was 6.5 times lower in SGA I infants than in AGA or SGA II infants (P < .001).

Conclusion: The SGA I infants appeared to have impaired utilization of circulating triglycerides, consistent with peripheral adipose depletion. Diminished fetus-derived apolipoprotein A-I levels with normal levels of placenta-derived apolipoprotein A-I levels might indicate a defect in the production or secretion of apolipoproteins associated with growth restriction.

Growth appears to be the best indicator of long-term fetal well-being and might be associated with development of abnormal states later in life.^1,2 Distinct from their appropriately grown counterparts, growth-restricted fetuses have physiologic characteristics that are believed to alter permanently the development and metabolic function of organs.³ In addition to increased morbidity and mortality, growth restriction has been associated neonatally with hypoglycemia, hypocalcemia, polycythemia, and thrombocytopenia.^4,5 Hypoinsulinemia, decreased thyrotropin levels, and diminished insulin-like growth factor–I have been described in small for gestational age (SGA) infants.⁶

Numerous confusing definitions have been used to describe infants and fetuses in the lower percentiles of growth. The term small for gestational age is used to describe infants and fetuses below the tenth percentile of growth for gestational age.⁷ In fetal growth restriction (FGR), an abnormal process limits the rate or extent of fetal growth, and application of the term aids in the identification of infants at risk for perinatal complications.^8,9 Although the terms relate to distinct processes, SGA and FGR often are used synonymously. Small for gestational age infants represent a mixed group of patients including constitutionally small, but otherwise normal, infants as well as growth-restricted infants. One method used to differentiate SGA infants is the ponderal index, which identifies infants with body habitus consistent with decreased peripheral fat deposition.¹⁰

Physiologic differences have been noted in growth-restricted infants. Intrauterine amino acid disturbances similar to the biochemical changes seen in postnatal protein-deprived states have been detected.¹¹ Protein metabolism defects and altered lipid metabolism also have been described. Studies^12,13 have suggested that deficiencies in essential fatty acids might be present in growth-restricted infants. Infusion of essential fatty acids in women with FGR produced a marked gain in fetal biparietal diameter and estimated weight.¹³ Some studies^14,15 showed SGA infants to be hypertriglyceridemic; however, those cases were linked with hypoxemia and hypoinsulinemia.¹⁶

Specific changes in lipid metabolism are associated with pregnancy. Normal plasma triglyceride, low-density lipoprotein cholesterol, and total cholesterol levels increase throughout pregnancy, with significantly elevated values after week 25. On the basis of those observations, apolipoprotein levels were determined in cord blood, and factors affecting lipid and lipoprotein levels were reviewed.^17,18 The high-density lipoprotein (HDL)/apolipoprotein A and apolipoprotein B/apolipoprotein A ratios were found to be greater in the FGR group, primarily because of a decrease in apolipoprotein A level.

In this study, we identified 38 infants below the tenth percentile for growth based on gestational age. Those infants were subdivided further into two groups based on ponderal index, to examine whether lipid metabolism differences could be detected between those two distinct subsets, comparing markers of lipid transport and metabolism.

	Materials and Methods

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The infants studied were delivered at either University of Louisville Hospital or Norton Hospital (Louisville, KY). Sera were collected from 25 appropriate for gestational age (AGA) infants and 38 infants identified as SGA. The AGA group involved term births between March 16 and 20, 1998, without perinatal complications. The 38 SGA infants subsequently were divided into two distinct groups based on ponderal index. The ponderal index was computed as follows: [(weight in grams) ÷ (crown-to-heel length in centimeters)³] x 100. Umbilical cord arterial blood specimens were collected in standard red-top collection tubes at the time of delivery. The blood samples were allowed to clot and then were centrifuged at 400 x g for 10 minutes. The serum was removed and stored at -20C until analysis.

To measure the total lipid concentration in the serum samples, serum (0.5 mL) was extracted by a modified procedure of Bligh and Dyer,¹⁹ using a two-step extraction with chloroform:methanol:water. The lipids from the serum were concentrated by drying under nitrogen gas at 4C, the serum lipid fractions were weighed, and the three groups were compared.

Subsequently, 500 µg of lipid extracted from each sample was separated by thin-layer chromatography, using a silica gel G plate and a mobile phase of chloroform:methanol:water (1:1:0.2). After separation, the lipid spots were visualized using iodine vapor, and those corresponding to phospholipids, free fatty acids, diglycerides, and triglycerides (based on simultaneously run standards) were scraped from the plate. After elution, each lipid group was quantitated by weighing and the mean (± standard deviation [SD]) of duplicate samples was calculated.

Apolipoprotein A-I levels were analyzed by western immunoblot. Albumin was removed from serum by absorption with Affi-Gel Blue (Bio-Rad, Richmond, CA). Serum proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions on a 14% acrylamide gel by the method of Laemmli²⁰ and were transferred electrophoretically onto nitrocellulose membranes for western immunoblot analysis.²¹ The blots were probed with rabbit anti–apolipoprotein A-I polyclonal antiserum (Accurate Scientific, Union, NY). The membranes subsequently were incubated with swine anti-rabbit immunoglobulin G, conjugated with horseradish peroxidase. The bound complexes were viewed by enhanced chemiluminescense (ECL; Amersham Life Sciences, Arlington Heights, IL) and immunoreactive bands were compared with prestained molecular weight standards for determination of molecular weight. The levels of the 22- and 28-kD apolipoprotein A-I bands were quantitated by densitometry.

To determine whether the differences observed were statistically significant, mean and SD values for the various components were determined for each group. With the use of InStat software (Graph-Pad, San Diego, CA), differences in values between controls and the SGA groups were evaluated by analysis of variance, with Tukey-Kramer multiple comparisons tests applied when there were significantly different results. The correlation of preeclampsia, smoking, and low ponderal indices was analyzed by Fisher exact test.

	Results

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The control group consisted of 25 AGA infants born at term. Small for gestational age infants were divided into two groups, based on ponderal index: the SGA I group consisted of infants with a ponderal index below the tenth percentile and SGA II infants had a ponderal index above the tenth percentile. The mean gestational age of the control group was 39.2 ± 1.1 weeks, whereas the mean for SGA I infants was 37.2 ± 3.0 weeks and for SGA II infants was 37.6 ± 2.5 weeks. Statistical analysis of gestational age indicated a significant difference between the three groups (analysis of variance, P = .009); however, only the comparison of the SGA I group with the control group yielded a statistical difference (P < .05). There were no significant differences in gestational ages between the SGA I and SGA II groups or between the SGA II and control groups.

The mean (± SD) birth weight for the control group was 3509.6 ± 320.5 g. The mean birth weight for SGA I infants was 2075.3 ± 530.7 g and for SGA II infants was 2270.7 ± 336.4 g. Differences in birth weights were highly significant among the groups (P < .001), with birth weights in the SGA I and SGA II groups both different from those in the control group (P < .001).

Charts of infants and mothers in the SGA groups were reviewed for differences in maternal smoking, presence of hypertensive disorders, oligohydramnios, and prolonged or complicated neonatal hospital course. Twelve of 20 SGA I mothers had hypertensive complications, compared with six of 18 SGA II mothers (P = .15). Eight of 12 SGA I hypertensive women had preeclampsia or eclampsia, whereas only three of six SGA II mothers had preeclampsia. The remainder of the complications were gestational hypertensions. Only one of 25 controls was preeclamptic (P = .006 versus SGA I mothers). Only one pregnancy was complicated by oligohydramnios, and that pregnancy was in the SGA I group. There were no statistical differences in prevalence of smoking or neonatal course among the three groups. One neonatal death occurred in the SGA II group; however, the infant was determined postmortem to have severe congenital central nervous system anomalies, not diagnosed antenatally.

Sera from control infants had a mean (± SD) total lipid content of 526 ± 198 µg/mL, compared with the SGA I group, which had a mean serum total lipid concentration of 205 ± 182 µg/mL, and the SGA II group, which had a mean concentration of 173 ± 82 µg/mL (2.8 times higher). The difference between the two SGA groups and controls was statistically significant (P < .001). The difference in levels of total lipid between the SGA I and SGA II groups was not significant. Regression analysis showed no discernible relationship within the SGA groups between total lipid concentrations and gestational age or birth weight.

Differences in the composition of serum lipids between groups are shown in Figure 1. Free fatty acid, triglyceride, diglyceride, and phospholipid levels were analyzed and values were compared between groups. All were expressed as percentages of total serum lipids. No significant differences were detected in the diglyceride or phospholipid populations between groups. However, significant differences were present in the free fatty acid and triglyceride fractions.

View larger version (27K): [in this window] [in a new window]   Figure 1. Distribution of cord blood–derived lipids (500 µg) as free fatty acids, phospholipids, triglycerides, and diglycerides in control infants (appropriate for gestational age), small for gestational age (SGA) I infants (ponderal index below the tenth percentile) and SGA II infants (ponderal index above the tenth percentile), as percentages of total serum lipids.

The distribution of serum lipids in control and SGA II infants was similar, with only the difference in triglyceride concentrations being statistically significant (P < .05) (1.3 times higher). In terms of relative percentages of total lipids, triglycerides were elevated and free fatty acids were decreased in the SGA I group, as compared with controls and the SGA II group, and these differences were statistically significant (P < .001 for both comparisons). In terms of absolute lipid values, both free fatty acid and triglyceride levels were significantly decreased in SGA I infants, compared with the AGA group (P < .001). The differences in those absolute values between SGA I (elevated triglyceride and decreased free fatty acid levels) and SGA II infants remained statistically significant (P < .01).

Studies showed 22-kD (placenta-derived) and 28-kD (fetal liver–derived) forms of apolipoprotein A-I to be associated with fetal tissues.¹⁸ The comparison of relative quantities of those apolipoprotein A-I proteins is presented in Figure 2. Quantitation with the bands resulting from densitometric analysis indicated similar levels of 22-kD apolipoprotein A-I between groups; however, a significant difference was observed between the levels of 28-kD apolipoprotein A-I in SGA I versus control or SGA II infants (P < .001). The SGA I group exhibited a decrease of approximately 6.5 times in the level of fetal liver–derived apolipoprotein A-I, compared with controls.

View larger version (21K): [in this window] [in a new window]   Figure 2. Levels of placenta-derived apolipoprotein A-I (22 kD) and fetal liver–derived apolipoprotein A-I (28 kD) in the sera obtained from control (appropriate for gestational age), small for gestational age (SGA) I (ponderal index below the tenth percentile), and SGA II (ponderal index above the tenth percentile) infants. Apolipoprotein A-I levels were analyzed by western immunoblotting and quantitated by densitometry.

	Discussion

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The results of this study showed the heterogeneity of those infants labeled as SGA. The lipid profile of the SGA I group indicated markedly diminished fetal liver–derived apolipoprotein A-I levels and decreased absolute total lipid levels; however, at relative concentrations, triglycerides were elevated and free fatty acids lowered. Those alterations are consistent with an inability to hydrolyze circulating triglycerides, leading to diminished peripheral adipose deposition. The lower free fatty acid levels in the absence of adequate peripheral adipose deposition might be consistent with depletion secondary to use as an alternate fuel source or might be simply a reflection of a more severe degree of uteroplacental insufficiency in that group. The decreased liver-derived apolipoprotein A-I levels, with similar placental apolipoprotein A-I levels, suggest impaired hepatic synthesis or secretion, and the finding is consistent with reports of low HDL and limited ability to metabolize very low density lipoproteins and to clear intravenous lipids in SGA infants early in the neonatal period.¹⁷

A limitation of this study was the failure to examine separately the hypertensive group with regard to etiology of growth restriction. A significant percentage of the SGA I mothers were preeclamptic (eight of 20 versus one of 25 controls, P = .006). Three of 18 mothers in the SGA II group were preeclamptic (P = .29 versus controls). The expected placental vascular changes in those patients might explain the significantly decreased free fatty acid levels in the SGA I group. Hypertensive pregnancies and their effects on endogenous fetal lipid metabolism will be examined in future studies.

These results show that SGA infants with a ponderal index below the tenth percentile have cord serum lipid characteristics distinct from similarly small but appropriately proportioned infants (SGA II group). The lipid values of the SGA I group indicated a diminished ability to deposit serum triglycerides in sufficient amounts to provide adipose stores, diminished levels of available free fatty acids, and impaired formation or secretion of apolipoprotein A-I, all of which are critical in the transition to neonatal life. Because of the risk of fetal and neonatal death, and the link with other disease states later in life for infants in the lower ranges of fetal growth, it is essential to determine the nature of the factors leading to low birth weight. Infants in that broad classification show wide ranges of metabolic impairment, knowledge of which is important for optimal neonatal outcome.

	Footnotes

 
PII S0029-7844(98)00489-X

Received July 13, 1998. Received in revised form September 28, 1998. Accepted October 15, 1998.

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