The Journal of Nutrition Vol. 127 No. 10 October 1997, pp. 1989-1992
Copyright ©1997 by the American Society for Nutritional Sciences

The Fatty Acid Composition of Maternal Diet Affects Lung Prostaglandin E₂ Levels and Survival from Group B Streptococcal Sepsis in Neonatal Rat Pups^1,2,3

Jorge I. Rayon, Jane D. Carver⁴, Lance E. Wyble, Doris Wiener, Sonja S. Dickey^*, Valerie J. Benford, Li T. Chen, and Daniel V. Lim^*

Department of Pediatrics, Division of Neonatology, University of South Florida College of Medicine, Tampa FL 33606; ^* Department of Biology, College of Arts and Sciences, University of South Florida, Tampa FL 33620; and   Department of Anatomy, University of South Florida College of Medicine, Tampa FL 33612

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

LITERATURE CITED

ABSTRACT

Dietary fatty acid effects upon the immune system may be mediated in part by effects upon the synthesis of proinflammatory mediators. The effects of maternal dietary fatty acid composition upon lung prostaglandin (PG) E₂ levels and survival from group B streptococcal (GBS) infection were investigated in neonatal rat pups. Beginning on d 2 of gestation and throughout lactation, pregnant dams were fed a purified diet whose fat source (22% of energy) was either corn oil or menhaden fish oil. On postnatal d 3, pups were randomly cross-fostered to dams of the same diet group to minimize litter effects; litters were then culled to 10 pups per dam. On postnatal d 7, pups were either injected with 1 × 10^7.5 GBS organisms or were killed for determination of lung tissue levels of PGE₂ and lung and erythrocyte fatty acid composition. Arachidonic acid and PGE₂ levels were significantly higher in the lungs of pups in the corn oil group compared with the fish oil group. Forty-nine percent of pups in the corn oil group survived the GBS challenge compared with 79% of pups in the fish oil group (P = 0.0005). These data suggest that the fatty acid composition of pre- and/or postnatal diet affects the neonatal response to immune challenge, which may be due in part to effects upon the synthesis of pro-inflammatory mediators.

INTRODUCTION

Group B streptococcal (GBS)⁵ sepsis, the leading cause of early onset neonatal bacterial infection, is characterized by rapid onset of fulminant shock, pneumonia and meningitis. Mortality rates may be as high as 50% (Baker and Edwards 1995). Pulmonary lesions are the predominant pathology, with histologic evidence of pneumonia being present in >50% of patients with fatal early onset infection (Baker and Edwards 1995, Vollman et al. 1976). Hemodynamic sequelae of GBS sepsis include pulmonary hypertension, reduced cardiac output and systemic hypotension, hypoxemia, granulocyte trapping in the lungs and increased pulmonary vascular permeability (Baker and Edwards 1995, Hemming et al. 1984, Rojas and Stahlman 1984).

Increased blood and tissue levels of eicosanoids and cytokines play an important role in mediating the hemodynamic and inflammatory sequelae of GBS sepsis (Baker and Edwards 1995, Gibson et al. 1995, Ling et al. 1995). Group B streptococcal organisms invade the lung microvascular endothelium and induce severe lung injury via the release of vasoactive arachidonic acid [20:4(n-6)]-derived eicosanoids such as prostaglandin (PG) I₂, PGE₂ and thromboxane (TX) A₂ (Baker and Edwards 1995, Gibson et al. 1995), whereas increased levels of TXA₂ are reportedly responsible for sepsis-induced pulmonary hypertension (Baker and Edwards 1995, Sandberg 1994, Truog et al. 1986). Proinflammatory cytokines, potent vasoactive substances with a wide range of physiologic effects, also play an important role in mediating tissue damage during GBS sepsis (Mancuso et al. 1994b, Teti et al. 1992, Williams et al. 1993). Administration of inhibitors of eicosanoid and cytokine synthesis and action can prolong survival from GBS sepsis (Del Moral et al. 1996, Givner et al. 1995, Mancuso et al. 1994a, Teti et al. 1993).

Diets supplemented with (n-3) fatty acids are used to attenuate eicosanoid and cytokine synthesis (Blok et al. 1996, Caughey et al. 1996, Gallai et al. 1995, Meydani et al. 1993). In the present study, survival from GBS sepsis was studied in 7-d-old rat pups whose dams were fed a diet enriched with either (n-6) or (n-3) fatty acids throughout pregnancy and lactation. This model of GBS infection has been demonstrated to have physiologic effects that parallel neonatal infection (Baker and Edwards 1995, Zeligs et al. 1982).

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MATERIALS AND METHODS

Animals and diets

This protocol was approved by the University of South Florida Laboratory Animal Medicine Experimentation Committee.

Beginning on d 2 of gestation and throughout lactation Sprague Dawley rats (Zivic Miller, Pittsburg, PA) were fed a reference diet (Laboratory Rodent Diet 5001, Purina Mills, Richmond, Indiana)⁶ or a synthetic diet (Purina Basal Diet 5755)⁷ whose fat source was either corn oil or menhaden fish oil. Diets were stored at 20°C and contained 125 µg/g ethoxyquin to minimize oxidation. All diets provided 17.4 kJ/g. Dams were caged individually and had free access to diets and water. Fresh diet was provided every 48 h.

On postnatal d 3, pups were cross-fostered to dams of the same diet groups to minimize litter effects. Litters were then culled to 10 per dam. Milk was removed from culled pup stomachs and stored at 70°C. Remaining pups were used either for assessment of survival following GBS challenge or for analysis of lung PGE₂ levels and lung and erythrocyte fatty acid composition.

GBS challenge

GBS inoculum. GBS serotype I (strain USF 704, a clinical isolate from the placenta of a woman delivering a septic newborn) was streaked from a 70°C stock culture onto a sheep blood agar plate and incubated at 37°C in 5% CO₂ for 24 h. Isolated GBS colonies on the plate were resuspended in Todd-Hewitt broth to a concentration of ~10¹² cells/L. The Todd-Hewitt broth suspension was used to charge 1-mL sterile syringes with the appropriate number of bacteria for injection of 0.1 mL into pups. Viable counts were taken of the Todd-Hewitt bacterial suspension to determine the actual number of GBS organisms injected into pups and to ensure there was no contamination of the suspensions.

GBS challenge. On postnatal d 7, pups were injected intraperitoneally with a mean dose of 1 × 10^7.5 GBS colony-forming units. Mortality was assessed over the subsequent 48 h. Spleens were removed from pups who died and cultured for the presence of GBS.

Spleen cultures. Spleen suspensions were prepared in cold 1× PBS (Gibco BRL, Grand Island, NY) using a Tekmar stomacher 80 lab blender (Cincinnati, OH). Suspensions were aseptically streaked onto sheep blood agar plates, incubated at 37°C in 5% CO₂ for 24 to 48 h, and examined for the presence of GBS; -hemolytic colonies were identified as GBS using the Phadebact Strep B Test (Boule Diagnostics AB, Huddinge, Sweden).

Lung prostaglandin E₂ levels and lung and erythrocyte fatty acid composition

Lung and erythrocytes. On postnatal d 7, pups were killed using a lethal dose of sodium pentobarbital (75 mg/kg, intraperitoneally). Lungs were quickly removed and snap-frozen in liquid nitrogen, and blood was collected by cardiac puncture. Lungs and erythrocytes were stored at 70°C. Lung homogenates were prepared on crushed ice using cold 0.05 mol/L Tris, 0.001 mol/L EDTA.

Lung prostaglandin E₂ analyses. To determine PGE₂ recoveries, lung homogenates were spiked with [³H]PGE₂ (NEN DuPont, Boston, MA) at 2.9 MBq/L homogenate. Homogenates were acidified with 2 mol/L HCl (pH 3.0), and prostaglandins were extracted three times with cold ethyl acetate. Percent recoveries were determined by counting ³H in pooled extracts. The manufacturer of the enzyme immunoassay kit used for PGE₂ determinations (Cayman Chemical, Ann Arbor, MI) reports 100, 43 and 19% specificity for PGE₂, PGE₃ and PGE₁, respectively. Therefore, PGE₂ from extracts was isolated by HPLC using a reverse-phase C-18 column and a mobile phase of 0.05 mol/L acetic acid-acetonitrile (690:310), pH to 5.8 with triethylamine. Column effluents were monitored at 220 nm. Recovered PGE₂ fractions, as determined by the presence of [³H]PGE₂, were pooled, dried and quantified using the enzyme immunoassay kit. Percent recoveries were again determined by counting ³H in column effluents.

Fatty acid analysis. All solvents were glass distilled, with 50 mg/L of butylated hydroxytoluene added to extraction solvents to minimize auto-oxidation. Lipid extracts of diet, maternal milk, pup lung and pup erythrocytes were prepared, methylated (Carlson et al. 1986), layered with nitrogen and analyzed within 24 h of extraction. Fatty acid methyl esters were separated and quantified with a Hewlett Packard (Palo Alto, CA) Series II gas chromatograph attached to a Hewlett Packard 3396A integrator and Hewlett Packard 7673A controller. Analyses were made using a 50-m capillary SP2330 column (Supelco Inc., Bellefonte, PA). Initial column temperature of 50°C was increased by 20°C/min to 170°C, then by 2°C/min to a final temperature of 220°C. Identification of individual fatty acid methyl esters was made by comparison of their retention times with those of authentic standards (NuChek Prep, Elysian MN, or Supelco, Inc.).

Protein analysis. Protein content of lung homogenates was measured using a BCA kit (Sigma Chemical, St. Louis, MO).

Statistical analyses. Statistical analyses were performed using the SPSS Statistical Package, Release 7 (SPSS, Chicago, IL); comparisons were made between the fish oil and corn oil groups only. Chi-square analysis was used to determine differences in mortality rates, with each pup considered an n of one. Levene's test for equality of variances and Student's t test were used to determine differences in body weights, length of gestation, fatty acid compositions and lung PGE₂ levels. P values less than 0.05 were considered significant. Values are reported as means ± SD.

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RESULTS

Nine dams were fed each of the three diets, yielding 90 pups per diet group after cross-fostering and culling to 10 pups per dam. Eight litters (80 pups) per diet group were used for the GBS mortality study, and one litter (10 pups) per diet group was used for analysis of lung PGE₂ levels and lung and erythrocyte fatty acid composition. Length of gestation and dam and pup weights did not differ among diet groups.

Fatty acid composition. The fish oil-supplemented diet contained substantial quantities of (n-3) fatty acids with more than 20 carbons, whereas the corn oil-supplemented diet had no n-6 or n-3 fatty acids with more than 20 carbons (Table 1). The fatty acid composition of maternal milk and pup lungs and erythrocytes reflected the maternal diet, with significant enrichment of (n-6) and (n-3) fatty acids in the corn oil and fish oil groups, respectively (Table 2). Levels of 20:4(n-6) were 68% higher in lungs of pups of the corn oil group, whereas levels of eicosapentanoic acid [20:5(n-3)] were 96% lower (P < 0.01).

Table 1. Fatty acid composition of diets fed to rats throughout pregnancy and lactation [View Table]

Table 2. Fatty acid composition of maternal milk of rats fed fish oil or corn oil supplemented diet and of the erythrocytes and lungs of their pups1,2 [View Table]

Lung prostglandin E₂. Mean recovery of [³H]PGE2 was approximately 80%. The PGE₂ levels in the lungs of pups in the fish oil and corn oil groups were 220 ± 106 and 760 ± 276 pg/mg protein, respectively (P = 0.004); levels in the reference group were 280 ± 122 pg/mg protein.

GBS sepsis survival. Mortality occurred within 48 h of GBS inoculation: 49% of pups in the corn oil group survived compared with 79% in the fish oil group (P = 0.0005); 67% of pups in the reference group survived.

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DISCUSSION

During infection, increased blood and tissue levels of eicosanoids influence immune cell activity and modulate hemodynamic sequelae. GBS induce severe lung injury via the release of 20:4(n-6)-derived vasoactive and inflammatory eicosanoids such as PGE₂, PGI₂ and TXA₂ (Baker and Edwards 1995, Gibson et al. 1995); interstitial inflammatory exudate is a consistent feature (Ablow et al. 1976). Thromboxane A₂ metabolites are found in high concentrations in lung lymph during GBS sepsis, and TXA₂ is reported to be responsible for sepsis-induced pulmonary hypertension (Baker and Edwards 1995, Sandberg et al. 1994). Treatment with inhibitors of eicosanoid synthesis can reduce the increase in pulmonary and systemic vascular resistance, improve cardiac output, stroke volume, lung compliance and oxygenation, and attenuate GBS sepsis-associated pulmonary hypertension (Baker and Edwards 1995, Gibson et al. 1995, Sandberg et al. 1994, Truog et al. 1986).

Increased synthesis of proinflammatory cytokines during infection can enhance host defense and modulate normal tissue remodeling. However, excessively high concentrations contribute to tissue injury and septic shock (Blok et al. 1996, Tracey et al. 1986). Tumor necrosis factor (TNF )  levels are particularly elevated during GBS sepsis (Baker and Edwards 1995, Ling et al. 1995, Teti et al. 1993), and the administration of TNF monoclonal antibodies or substances that inhibit TNF release can attenuate the inflammatory and cardiovascular manifestations of GBS sepsis and reduce mortality (Del Moral et al. 1996, Givner et al. 1995, Mancuso et al. 1994a, Teti et al. 1993).

Diets supplemented with fish oils are used to attenuate eicosanoid and cytokine synthesis because these diets result in replacement of 20:4(n-6) with 20:5(n-3) in membrane phospholipids (Blok et al. 1996, Caughey et al. 1996, Gallai et al. 1995, Meydani et al. 1993). Eicosanoids derived from 20:5(n-3) have less inflammatory activity and different vasoactive effects compared with those derived from 20:4(n-6) (Blok et al. 1996, Zurier 1993); 20:5(n-3) may also exhibit antiinflammatory effects by inhibiting the synthesis of leukotrienes (Lee et al., 1985). Although results vary depending upon species and tissue examined, fish oil-supplemented diets are also associated with lowered production of proinflammatory cytokines, particularly in humans (Blok et al. 1996, Gallai et al., 1995). These effects may be mediated through modulation of cytokine gene expression (Robinson et al. 1995), by alterations in eicosanoid regulation of cytokine synthesis, or by effects upon intracellular transduction pathways involved in cytokine synthesis (Blok et al. 1996). In the present study, lower mortality among pups of the fish oil-fed group may have been due in part to lower blood and tissue levels of eicosanoids derived from 20:4(n-6), as suggested by lower levels of PGE₂ in pup lungs.

In summary, neonatal rat pups of dams fed corn oil-supplemented diet throughout pregnancy and lactation had higher erythrocyte and lung tissue levels of (n-6) fatty acids, higher mortality following GBS challenge and higher lung PGE₂ levels compared with pups of dams fed a fish oil-supplemented diet. These data suggest that the fatty acid composition of pre- and/or postnatal diet affects response to neonatal immune challenge and that this effect may be due in part to alterations in the synthesis of pro-inflammatory mediators.

FOOTNOTES

Manuscript received 3 February 1997. Initial reviews completed 28 March 1997. Revision accepted 4 June 1997.

LITERATURE CITED

• Ablow R. C., Driscoll S. G., Effmann E. L., Effmann E. L., Gross I., Jolles C. J., Uauy R., Warshaw J. B. A comparison of early-onset group B streptococcal neonatal infection and the respiratory distress syndrome of the newborn. N. Engl. J. Med. 1976; 294:65-70 [Abstract]
• Baker, C. J. & Edwards, M. S. (1995) Group B streptococcal infections. In: Infectious Diseases of the Fetus and Newborn (Remington, J. S. & Klein, J. O., ed.), pp. 980-1054. W.B. Saunders Co., Philadelphia, PA.
• Blok W. L., Katan M. B., Van Der Meer J.W.M. Modulation of inflammation and cytokine production by dietary (n-3) fatty acids. Critical review. J. Nutr. 1996; 126:1515-1533
• Carlson S., Carver J., House S. High fat diets varying in ratios of polyunsaturated to saturated fatty acid and linoleic to linolenic acid: a comparison of rat neural and red cell membrane phospholipids. J. Nutr. 1986; 116:718-725
• Caughey G. E., Mantzioris E., Gibson R. A., Cleland L. G., James M. J. The effect on human tumor necrosis factor  and interleukin 1 production of diets enriched in n-3 fatty acids from vegetable oil or fish oil. Am. J. Clin. Nutr. 1996; 63:116-122 [Abstract/Free Full Text]
• Del Moral T., Goldberg R. N., Urbon J., Suguihara C., Martinez O., Stein-Streilein J., Feuer W. J., Bancalari E. Effects of treatment with pentoxifylline on the cardiovascular manifestations of group B streptococcal sepsis in the piglet. Pediatr. Res. 1996; 40:469-473 [Medline]
• Gallai V., Sarchielli P., Trequattrini A., Franceschini M., Floridi A., Firenze C., Alberti A., DiBenedetto D., Stragliotto E. Cytokine secretion and eicosanoid production in the peripheral blood mononuclear cells of MS patients undergoing dietary supplementation with n-3 polyunsaturated fatty acids. J. Neuroimmunol. 1995; 56:143-153 [Medline]
• Gibson R. L., Solderland C., Henderson W. R., Chi E. Y., Rubens C. E. Group B streptococci (GBS) injure lung endothelium in vitro: GBS invasion and GBS-induced eicosanoid production is greater with microvascular than with pulmonary artery cells. Infect. Immun. 1995; 63:271-279 [Abstract]
• Givner L. B., Gray L., O'Shea T. M. Antibodies to tumor necrosis factor-: use as adjunctive therapy in established group B streptococcal disease in newborn rats. Pediatr. Res. 1995; 38:551-554 [Medline]
• Hemming V. G., O'Brien W. F., Fischer G. W., Golden S. M., Noble S. F. Studies of short-term pulmonary and peripheral vasculature responses induced in oophorectomized sheep by the infusion of a group B streptococcal extract. Pediatr. Res. 1984; 18:266-269 [Medline]
• Lee T. H., Hoover R. L., Williams J. D., Sperling R. I., Ravalese J. III, Spur B. W., Robinson D. R., Corey E. J., Lewis R. A., Austen K. F. Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function. N. Engl. J. Med. 1985; 312:1217-1224 [Abstract]
• Ling E.W.Y., Noya F.J.D., Ricard G., Beharry, K. Mills, E. L., Aranda J. V. Biochemical mediators of meningeal inflammatory response to group B streptococcus in the newborn piglet model. Pediatr. Res. 1995; 38:981-987 [Medline]
• Mancuso G., Cusumano V., Cook J. A., Smith E., Squadrito F., Balndin G., Teti G. Efficacy of tumor necrosis factor alpha and eicosanoid inhibitors in experimental models of neonatal sepsis. FEMS Immunol. Med. Microbiol. 1994a; 9:49-54 [Medline]
• Mancuso G., Tomasello F., von Hunolstein C., Orefici G., Teti G. Induction of tumor necrosis factor alpha by the group- and type-specific polysaccharides from type III group B streptococci. Infect. Immun. 1994b; 62:2748-2753 [Abstract/Free Full Text]
• Meydani S. N., Lichtenstein A. H., Cornwall S., Meydani M., Goldin B. R., Rasmussen H., Dinarello C. A., Schaefer E. J. Immunologic effects of national cholesterol education panel step-2 diets with and without fish-derived n-3 fatty acid enrichment. J. Clin. Invest. 1993; 92:105-113
• Robinson, D. R., Urakaze M., Huang, R., Taki, H., Sugiyama, E. Knoell, C. T., Xu, L., Yeh, E.T.H. & Auron, P. E. (1995) Dietary marine lipids suppress the continuous expression of interleukin 1 gene transcription. Proceedings from the 2nd International Congress of the International Society for the Study of Fatty Acids and Lipids, Bethesda, MD.
• Rojas J., Stahlman M. The effects of group B streptococcus and other organisms on the pulmonary vasculature. Clin. Perinatol. 1984; 11:591-599 [Medline]
• Sandberg K., Edberg K. E., Fish W., Parker R. A., Hellerqvist C., Sundell H. Thromboxane receptor blockade (SQ 29,548) in group B streptococcal toxin challenge in young lambs. Pediatr. Res. 1994; 35:571-579 [Medline]
• Teti G., Mancuso G., Tomasello F., Chiofalo M. S. Production of tumor necrosis factor- and interleukin-6 in mice infected with group B streptococci. Circ. Shock 1992; 38:138-144 [Medline]
• Teti G., Mancuso G., Tomasello F. Cytokine appearance and effects of anti-tumor necrosis factor alpha antibodies in a neonatal rat model of group B streptococcal infection. Infect. Immun. 1993; 61:227-235 [Abstract/Free Full Text]
• Tracey K. J., Beutler B., Lowry S. F., Merryweather J., Wolpe S., Milsark I. W., Harri R. J., Fahey T. J. III, Zentella A., Albert J. D., Shires G. T., Cerami A. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234:470-474 [Abstract/Free Full Text]
• Truog W. E., Sorensen G. K., Standaert T. A., Redding G. J. Effects of the thromboxane synthetase inhibitor, dazmegrel (UK 38,485), on pulmonary gas exchange and hemodynamics in neonatal sepsis. Pediatr. Res. 1986; 20:481-486 [Medline]
• Vollman J. H., Smith W. L., Ballard E. T., Light I. J. Early onset group B streptococcal disease: clinical, roentgenographic, and pathologic features. J. Pediatr. 1976; 89:199-203 [Medline]
• Williams P. A., Bohnsack J. F., Augustine N. H., Drummond W. K., Rubens C. E., Hill H. R. Production of tumor necrosis factor by human cells in vitro and in vivo, induced by group B streptococci. J. Pediatr. 1993; 23:292-300
• Zeligs B. J., Armstrong C. D., Walser J. B., Bellanti J. A. Age-dependent susceptibility of neonatal rats to group B streptococcal type III infection: correlation of severity of infection and response of myeloid pools. Infect. Immun. 1982; 37:255-263 [Abstract/Free Full Text]
• Zurier R. B. Fatty acids, inflammation and immune responses. Prostaglandins Leukotrienes Essent. Fatty Acids 1993; 48:57-62 [Medline]

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