Polyunsaturated Fatty Acid Intake during Complementary Feeding and Neurodevelopmental Outcome in Very Low Birth Weight Infants.

Polyunsaturated fatty acids (PUFAs) are vital for brain development, yet limited knowledge exists regarding PUFA intake during complementary feeding (CF) and its impact on neurodevelopmental outcomes in very low birth weight (VLBW) infants. This secondary analysis of a randomized intervention trial, aimed to investigate the association between dietary intake of total PUFAs, arachidonic acid (AA), and docosahexaenoic acid (DHA) during CF and neurodevelopmental outcomes at 12 and 24 months of corrected age (CA). Dietary intakes were assessed using monthly 3 day dietary protocols from 3 to 12 months CA. Neurodevelopmental outcome was evaluated using the Bayley Scales of Infant Development-III. Among the 177 randomized patients, PUFA intake and neurodevelopmental outcomes were evaluated in 140 (79%) infants. Higher total PUFA and DHA intakes significantly correlated with improved cognitive and motor function at 12 months CA, while increased AA intake notably enhanced motor scores at 12 months CA. However, median dietary intakes of AA and DHA (AA: 53.50-84.25 mg/d; DHA: 51.47-76.23 mg/d) fell short of recommended levels (AA: 140 mg/d; DHA: 100 mg/d) at any of the investigated timepoints. These findings emphasize the need to enhance total PUFA, DHA and AA intakes during CF, ensuring adherence to guidelines and unlocking the potential to improve neurodevelopmental outcomes in VLBW infants.

Effects of early introduction of solid foods on nutrient intake in preterm infants during their 1st year of life: a secondary outcome analysis of a prospective, randomized intervention study.

Very low birth weight (VLBW) infants have higher nutritional needs even after hospital discharge. However, data concerning current nutrient intakes at different time points after the introduction of solid foods and whether dietary reference values are being met are scarce. To adress this issue, this secondary analysis of a prospective, two-arm interventional study in 177 VLBW infants 21 investigates dietary intake comparing early and late (early: 10-12 weeks corrected for gestational age, late: 16-18 weeks corrected for gestational age) introduction of standardized complementary food during the first year of life. Nutritional intake was assessed using self-reported monthly 3-day dietary records from 3 until 12 months, corrected for gestational age. The time point of the introduction of solid foods did not influence nutrient intake, but the early introduction of solids tended toward a higher proportional intake of protein and carbohydrates and a lower intake of fat as a percentage of total energy) during the 1st year of life, corrected for gestational age. The results of this study indicate that this standardized feeding concept was sufficient for zinc, calcium, and phosphorus intake. However, dietary iron and vitamin D intakes did not meet the recommendations. Thus, prolonged iron supplementation should be considered beyond the introduction of meat and vitamin D supplementation at least until 12 months, corrected for gestational age. Trial registration number: ClinicalTrials.gov: NCT01809548.

A secondary Outcome Analysis of a Randomized Trial Using a Mixed Lipid Emulsion Containing Fish Oil in Infants with Extremely Low Birth Weight: Cognitive and Behavioral Outcome at Preschool Age.

OBJECTIVE: To evaluate the impact of a parenteral lipid emulsion containing fish oil compared with a soybean oil based-lipid emulsion on the cognitive outcome and behavior of preschool children with extremely low birth weight. STUDY DESIGN: This was a retrospective secondary outcome analysis of a randomized controlled trial performed between June 2012 and June 2015. Infants with extremely low birth weight received either a mixed (soybean oil, medium chain triglycerides, olive oil, fish oil) or a soybean oil-based lipid emulsion for parenteral nutrition. Data from the Kaufman Assessment Battery for Children II, the Child Behavior Checklist 1.5-5, and anthropometry were collected from medical charts at 5.6 years of age. RESULTS: At discharge, 206 of the 230 study participants were eligible. At 5 years 6 months of age, data of 153 of 206 infants (74%) were available for analysis. There were no significant differences in Kaufman Assessment Battery for Children II scores for Sequential/Gsm, Simultaneous/Gv, Learning/Glr, and Mental Processing Index (mixed lipid: median, 97.5 [IQR, 23.5]; soybean oil: median, 96 [IQR, 19.5]; P = .43) or Child Behavior Checklist 1.5-5 scores for internalizing problems, externalizing problems, or total problems (mixed lipid: median, 37 [IQR, 12.3]; soybean oil: median, 37 [IQR, 13.5]; P = .54). CONCLUSIONS: A RandomForest machine learning regression analysis did not show an effect of type of lipid emulsion on cognitive and behavioral outcome. Parenteral nutrition using a mixed lipid emulsion containing fish oil did not affect neurodevelopment and had no impact on child behavior of infants with extremely low birth weights at preschool age. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01585935.

Preterm Infants on Early Solid Foods and Vitamin D Status in the First Year of Life-A Secondary Outcome Analysis of a Randomized Controlled Trial.

Preterm birth places infants at high risk for mineral and micronutrient deficiencies important for bone health. The aim of this study was to examine whether two timepoints for the introduction of solid foods in preterm infants have an impact on vitamin D status in the first year of life. This is a secondary outcome analysis of a prospective, randomized trial on very low birth weight (VLBW) infants, randomized to an early (10-12th week corrected age) or a late (16-18th week corrected age) complementary-feeding group. Vitamin D status was assessed by blood samples taken at 6 weeks, 6, and 12 months corrected age. In total, 177 infants were randomized (early group: n = 89, late group: n = 88). There was a tendency toward lower levels of serum 25-OH-vitamin D in the early group throughout the first year of life (p = not significant (n.s.)); no differences were detected in the other parameters. At 6 months corrected age, infants of the early group had a significantly higher incidence of vitamin D deficiency. The timepoint of the introduction of solid foods had no impact on the serum 25-OH-vitamin D levels and other parameters important for bone health but showed a tendency toward lower levels in the early-feeding group.

Preterm Infants on Early Solid Foods and Iron Status in the First Year of Life-A Secondary Outcome Analysis of a Randomized Controlled Trial.

Introduction of solid foods and iron status in the first year of life of preterm infants are highly discussed topics. The aim of this study was to examine whether two timepoints of introduction of standardized solid foods in preterm infants have an impact on ferritin and other hematologic parameters important for iron status in the first year of life. This is a secondary outcome analysis of a prospective, randomized intervention trial in very low birth weight (VLBW) infants randomized to an early (10-12th week corrected age) or a late (16-18th week corrected age) complementary feeding group. Iron status was assessed with blood samples taken at 6 weeks, 6 months, and 12 months corrected age. In total, 177 infants were randomized (early group: n = 89, late group: n = 88). Ferritin showed no differences between study groups throughout the first year of life, as did all other parameters associated with iron status. At 12 months corrected age, the incidence of iron deficiency was significantly higher in the early feeding group. There is room for improvement of iron status in VLBW preterm infants, regular blood checks should be introduced, and current recommendations may need to be a reconsidered.

Neurodevelopmental Outcome of Extremely Low Birth Weight Infants with Cholestasis at 12 and 24 Months.

INTRODUCTION: The aims of the study were to describe the neurodevelopmental outcome of extremely low birth weight (ELBW) infants with parenteral nutrition-associated cholestasis (PNAC) and to assess whether PNAC is associated with adverse neurodevelopmental outcome. METHODS: The study is a secondary analysis of controlled trial (June 2012-October 2017) on PNAC incidence in ELBW infants receiving two different parenteral lipid emulsions (mixed lipid emulsion containing fish oil vs. soybean oil-based). Neurodevelopmental follow-up at 12- and 24-month corrected age was compared in infants with and without PNAC. A machine learning-based regression analysis was used to assess whether PNAC was associated with adverse neurodevelopmental outcome. RESULTS: For assessment of neurodevelopmental outcome (Bayley-III), 174 infants were available at 12-month (PNAC: n = 21; no PNAC: n = 153) and 164 infants at 24-month (PNAC: n = 20; no PNAC: n = 144) corrected age. The neurodevelopment of ELBW infants with PNAC was globally delayed, with significantly lower cognitive, language, and motor scores at both 12- and 24-month corrected age. Regression analyses revealed that PNAC was associated with an adverse motor outcome. CONCLUSION: ELBW infants with PNAC are at increased risk for adverse neurodevelopmental outcome.

Bet v 1 triggers antiviral-type immune signalling in birch-pollen-allergic individuals.

BACKGROUND: In allergic patients, clinical symptoms caused by pollen remind of symptoms triggered by viral respiratory infections, which are also the main cause of asthmatic exacerbations. In patients sensitized to birch pollen, Bet v 1 is the major symptom-causing allergen. Immune mechanisms driving Bet v 1-related responses of human blood cells have not been fully characterized. OBJECTIVE: To characterize the immune response to Bet v 1 in peripheral blood in patients allergic to birch pollen. METHODS: The peripheral blood mononuclear cells of birch-allergic (n = 24) and non-allergic (n = 47) adolescents were stimulated ex-vivo followed by transcriptomic profiling. Systems-biology approaches were employed to decipher disease-relevant gene networks and deconvolution of associated cell populations. RESULTS: Solely in birch-allergic patients, co-expression analysis revealed activation of networks of innate immunity and antiviral signalling as the immediate response to Bet v 1 stimulation. Toll-like receptors and signal transducer transcription were the main drivers of gene expression patterns. Macrophages and dendritic cells were the main cell subsets responding to Bet v 1. CONCLUSIONS AND CLINICAL RELEVANCE: In birch-pollen-allergic patients, the activated innate immune networks seem to be, in part, the same as those activated during viral infections. This tendency of the immune system to read pollens as viruses may provide new insight to allergy prevention and treatment.

A Mixed-Lipid Emulsion Containing Fish Oil for the Parenteral Nutrition of Preterm Infants: No Impact on Visual Neuronal Conduction.

Fish oil is rich in omega-3 fatty acids and essential for neuronal myelination and maturation. The aim of this study was to investigate whether the use of a mixed-lipid emulsion composed of soybean oil, medium-chain triglycerides, olive oil, and fish oil (SMOF-LE) compared to a pure soybean oil-based lipid emulsion (S-LE) for parenteral nutrition had an impact on neuronal conduction in preterm infants. This study is a retrospective matched cohort study comparing preterm infants <1000 g who received SMOF-LE in comparison to S-LE for parenteral nutrition. Visual evoked potentials (VEPs) were assessed longitudinally from birth until discharge. The latencies of the evoked peaks N2 and P2 were analyzed. The analysis included 76 infants (SMOF-LE: n = 41 and S-LE: n = 35) with 344 VEP measurements (SMOF-LE: n= 191 and S-LE n = 153). Values of N2 and P2 were not significantly different between the SMOF-LE and S-LE groups. A possible better treatment effect in the SMOF-LE group was seen as a trend toward a shorter latency, indicating faster neural conduction at around term-equivalent age. Prospective trials and follow-up studies are necessary in order to evaluate the potential positive effect of SMOF-LE on neuronal conduction and visual pathway maturation.

A Randomized Trial of Parenteral Nutrition Using a Mixed Lipid Emulsion Containing Fish Oil in Infants of Extremely Low Birth Weight: Neurodevelopmental Outcome at 12 and 24 Months Corrected Age, A Secondary Outcome Analysis.

OBJECTIVE: To examine whether parenteral nutrition using a mixed lipid emulsion containing fish oil improves the neurodevelopmental outcomes of extremely low birth weight infants. STUDY DESIGN: The study is a secondary outcome analysis of a double-blind randomized trial of 230 extremely low birth weight infants performed at a single level IV neonatal care unit (Medical University Vienna; June 2012 to June 2015). Participants received either a mixed lipid emulsion composed of soybean oil, medium chain triglycerides, olive oil, and fish oil, or a soybean oil-based lipid emulsion for parenteral nutrition. Neurodevelopment of study participants was assessed at 12 and 24 months corrected age (August 2013 to October 2017) using the Bayley Scales of Infant-Toddler Development, third edition. RESULTS: At discharge, 206 of the 230 study participants were eligible. At 12 and 24 months corrected age, 174 of 206 (85%) and 164 of 206 (80%) infants were evaluated. At 12 months, there was no significant difference in cognitive (mixed lipid: median, 95 [IQR, 85-101]; soybean oil: median, 95 [IQR, 85-100]; P = .71), language (mixed lipid: median, 86 [IQR, 77-94], soybean oil: median, 89 [IQR, 79-94]; P = .48), or motor scores (mixed lipid: median, 88 [IQR, 76-94], soybean oil: median, 88 [IQR, 79-94]; P = .69). At 24 months, there was again no significant difference in cognitive (mixed lipid: median, 95 [IQR, 80-105], soybean oil: median, 95 [IQR, 90-105]; P = .17), language (mixed lipid: median, 89 [IQR, 75-97], soybean oil 89 [IQR, 77-100]; P = .54), and motor scores (mixed lipid: median, 94 [IQR, 82-103], soybean oil: median, 94 [IQR, 85-103]; P = .53). CONCLUSIONS: Parenteral nutrition using a mixed lipid emulsion containing fish oil did not improve neurodevelopment of extremely low birth weight infants at 12 and 24 months corrected age. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01585935.

A Mixed Lipid Emulsion Containing Fish Oil and Its Effect on Electrophysiological Brain Maturation in Infants of Extremely Low Birth Weight: A Secondary Analysis of a Randomized Clinical Trial.

OBJECTIVE: To assess whether parenteral nutrition for infants of extremely low birth weight using a mixed lipid emulsion that contains fish oil influences electrophysiological brain maturation. STUDY DESIGN: The study is a prespecified secondary outcome analysis of a randomized controlled trial of 230 infants of extremely low birth weight receiving a mixed (soybean oil, medium-chain triglycerides, olive oil, and fish oil; intervention) or a soybean oil-based lipid emulsion (control). The study was conducted at a single-level IV neonatal care unit (Medical University Vienna; June 2012 to October 2015). Electrophysiological brain maturation (background activity, sleep-wake cycling, and brain maturational scores) was assessed biweekly by amplitude-integrated electroencephalography (birth to discharge). RESULTS: A total of 317 amplitude-integrated electroencephalography measurements (intervention: n = 165; control: n = 152) from 121 (intervention: n = 63; control: n = 58) of 230 infants of the core study were available for analysis. Demographic characteristics were not significantly different. By 28 weeks of postmenstrual age, infants receiving the intervention displayed significantly greater percentages of continuous background activity. Total maturational scores and individual scores for continuity, cycling, and bandwidth were significantly greater. Maximum maturational scores were reached 2 weeks earlier in the intervention group (36.4 weeks, 35.4-37.5) compared with the control group (38.4 weeks, 37.1-42.4) (median, IQR; P < .001). CONCLUSIONS: Using a mixed parenteral lipid emulsion that contains fish oil, we found that electrophysiological brain maturation was accelerated in infants who were preterm. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01585935.

A Mixed Lipid Emulsion for Prevention of Parenteral Nutrition Associated Cholestasis in Extremely Low Birth Weight Infants: A Randomized Clinical Trial.

OBJECTIVES: To examine whether a mixed lipid emulsion reduces the incidence of parenteral nutrition associated cholestasis (PNAC) in extremely low birth weight (ELBW, <1000 g) infants. STUDY DESIGN: This double-blind randomized trial of 230 ELBW infants (June 2012-October 2015) was performed at a single level IV neonatal intensive care unit. Patients received either a mixed lipid emulsion composed of soybean oil, medium chain triglycerides, olive oil, and fish oil-(intervention) or a soybean oil-based lipid emulsion (control) for parenteral nutrition. The primary outcome measure was PNAC (conjugated bilirubin >1.5 mg/dL [25 µmol/L] at 2 consecutive measurements). The study was powered to detect a reduction of PNAC from 25% to 10%. RESULTS: Reasons for noneligibility of 274 infants screened were refusal to participate (n = 16), death (n = 10), withdrawal of treatment (n = 5), higher order multiples (n = 9), and parents not available for consent (n = 4). Intention to treat analysis was carried out in 223 infants (7 infants excluded after randomization). Parenteral nutrition associated cholestasis was 11 of 110 (10.1%) in the intervention and 18 of 113 (15.9%) in the control group (P = .20). Multivariable analyses showed no statistically significant difference in the intention to treat (aOR 0.428, 95% CI 0.155-1.187; P = .10) or per protocol population (aOR 0.457, 95% CI 0.155-1.347; P = .16). There was no statistically significant effect on any other neonatal morbidity. CONCLUSIONS: The incidence of parenteral nutrition associated cholestasis was not significantly reduced using a mixed lipid emulsion in ELBW infants. TRIAL REGISTRATION: ClinicalTrials.govNCT01585935.

Correction: Does Visceral Osteopathic Treatment Accelerate Meconium Passage in Very Low Birth Weight Infants?- A Prospective Randomized Controlled Trial.

[This corrects the article DOI: 10.1371/journal.pone.0123530.].

Clinical Experience With Numeta in Preterm Infants: Impact on Nutrient Intake and Costs.

BACKGROUND: A new "ready-to-use" triple-chamber container, Numeta (Baxter, Deerfield, IL), is available for preterm parenteral nutrition (PN) to provide nutrients according to the recommendations of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the European Society for Clinical Nutrition and Metabolism (ESPEN) Guidelines for Pediatric Parenteral Nutrition. We investigated the clinical application of Numeta compared with individualized PN in preterm infants (≤1.500 g) and evaluated the effects on nutrient intake, costs, and preparation time. MATERIALS AND METHODS: In a clinical observational study, prescriptions for preterm infants were performed with the new prescription software catoPAN (Cato Software Solutions, Becton Dickinson, Vienna, Austria). Individualized PN and Numeta prescriptions were mirrored, and nutrition content of the PNs was compared with each other and with ESPGHAN/ESPEN recommendations. Furthermore, costs and preparation time were assessed. RESULTS: In total, 374 PN solutions (>1000 g [n = 333]/≤1000 g [n = 41]) were analyzed. Protein intake with Numeta was significantly lower compared with individualized PN and did not meet the recommendations for infants <1500 g during the first day and the period of transition after birth. Energy intake was significantly higher with Numeta. The costs for Numeta preparations were €18 (about US$20) higher than for individualized PN. However, the preparation time/solution was 2 minutes faster with Numeta. CONCLUSION: Numeta is an alternative to individualized PN for infants >1000 g in the period of stable growth when enteral feedings have already started. Protein intake is significantly lower than in individualized PN solutions. Numeta is more expensive in comparison to individualized PN but saves human resources.

Does visceral osteopathic treatment accelerate meconium passage in very low birth weight infants?- A prospective randomized controlled trial.

BACKGROUND: To determine whether the complementary approach of visceral manipulative osteopathic treatment accelerates complete meconium excretion and improves feeding tolerance in very low birth weight infants. METHODS: This study was a prospective, randomized, controlled trial in premature infants with a birth weight <1500 g and a gestational age <32 weeks who received a visceral osteopathic treatment 3 times during their first week of life or no treatment. RESULTS: Passage of the last meconium occurred after a median of 7.5 days (95% confidence interval: 6-9 days, n = 21) in the intervention group and after 6 days (95% confidence interval: 5-9 days, n = 20,) in the control group (p = 0.11). However, osteopathic treatment was associated with a 8 day longer time to full enteral feedings (p = 0.02), and a 34 day longer hospital stay (Median = 66 vs. 100 days i.e.; p=0.14). Osteopathic treatment was tolerated well and no adverse events were observed. CONCLUSIONS: Visceral osteopathic treatment of the abdomen did not accelerate meconium excretion in VLBW (very low birth weight)-infants. However infants in the osteopathic group had a longer time to full enteral feedings and a longer hospital stay, which could represent adverse effects. Based on our trial results, we cannot recommend visceral osteopathic techniques in VLBW-infants. TRIAL REGISTRATION: Clinical trials.gov: NCT02140710.

Effect of fortifiers and additional protein on the osmolarity of human milk: is it still safe for the premature infant?

OBJECTIVES: The present guidelines of the American Academy of Pediatrics recommend that osmolarity not exceed 450 mOsm/kg (or approximately an osmolarity of 400 mOsm/L) for breast milk or infant formulae, to minimize the risk factors for necrotizing enterocolitis. A commercial protein supplement has been developed to meet special protein requirements (4.0-4.5 g · kg(-1) · day(-1)) of infants with a birth weight <1000 g. Because its effect on osmolarity has not been systematically studied, we characterized the effects of fortification on the osmolarity of human milk (HM). METHODS: Osmolarity of fresh and processed HM was measured at baseline, after fortification with a commercial HM fortifier and after further supplementation with additional protein increasing in 0.5-g steps up to 4.0 g. Measurements were performed immediately after adding fortifier and/or protein and after 24 hours. In addition, changes in osmolarity were determined after adding therapeutic additives such as iron, multivitamin supplement, and calcium-phosphorus capsules. RESULTS: Native HM samples (n = 84) had 297 mOsm/L, (median; 95% confidence interval 295-299 mOsm/L). Adding HM fortifier increased osmolarity up to 436 mOsm/L (95% confidence interval 431-441 mOsm/L). Additional protein supplementation increased osmolarity by 23.5 mOsm/L per 0.5-g step, up to a maximum of 605 mOsm/L. Pasteurization decreased osmolarity by 20-30 mOsm/L (P < 0.001), and storage for 24 hours slightly increased osmolarity (by 11.5 mOsm/L P = 0.0002). Therapeutic additives increased osmolarity up to 868 mOsm/L. CONCLUSIONS: Adding HM fortifier and additional protein to HM increased osmolarity to >400 mOsm/L and therefore above the recommended threshold. Because of the excessive increase in osmolarity combinations of HM + fortifier and additional protein should not be applied together with multivitamins or other additives.

A randomized, controlled trial of the effects of adding vitamin B12 and folate to erythropoietin for the treatment of anemia of prematurity.

BACKGROUND: Premature infants, especially those with birth weights of <1500 g, often suffer from anemia of prematurity and associated problems. Erythropoietin therapy is a safe effective way to prevent and to treat anemia of prematurity. We hypothesized that combined administration of vitamin B12 and folate with erythropoietin and iron would enhance erythropoietin-induced erythropoiesis. METHODS: In a randomized, controlled trial, 64 premature infants (birth weight: 801-1300 g) receiving erythropoietin and iron supplementation were assigned randomly to receive either vitamin B12 (3 microg/kg per day) and folate (100 microg/kg per day) (treatment group) or a lower dose of folate (60 microg/kg per day) (control group). RESULTS: During the 4-week observation period, vitamin B12 and folate enhanced erythropoietin-induced erythropoiesis significantly, as indicated by a 10% increase in red blood cell counts, compared with folate alone. Hemoglobin and hematocrit levels remained stable in the treatment group, whereas they decreased in the control group. Vitamin B12 levels in the treatment group increased over baseline and control values, whereas red blood cell folate levels were comparable between the groups. Subsequent analysis showed slight nonsignificant differences in baseline red blood cell count, hemoglobin level, hematocrit level, and mean corpuscular volume values, which must be addressed as a limitation. CONCLUSIONS: With the limitation of a slight imbalance in baseline data between the study groups, combined therapy with vitamin B12, folate, erythropoietin, and orally and intravenously administered iron seemed more effective in stimulating erythropoiesis among premature infants, compared with erythropoietin, iron, and low-dose folate alone. Additional trials are necessary to confirm these data.

Metabolism of 4-n-nonylphenol by non-modified and CYP1A1- and CYP1A2-transgenic cell cultures of tobacco.

The metabolism of 14C-4-n-nonylphenol (l4C-4-n-NP), as a model for the xenoestrogen nonylphenol, was investigated in three types of tobacco cell suspension cultures: one genetically non-modified culture (NT) and two cultures constitutively expressing human cytochrome P450 CYP1A1 or CYP1A2. With 1 mg l(-1) of 14C-4-n-NP and 24 h of incubation, the xenobiotic was transformed almost completely to glycosides. After glycosidic cleavage, 14C-4-n-NP and several primary metabolites of 4C-4-n-NP were liberated. Portions of the primary metabolites were 29.3% (NT culture), 34.3% (CYP1A1 culture), and 50.7% of applied 14C (CYP1A2 culture). Thus, the endogenous capacity of the tobacco cells to form primary metabolites of 4-n-NP was noticeably higher than that of CYP1A1 or CYP1A2. The results however clearly suggest that 4-n-NP is - even though a poor - substrate of CYP1A1 and CYP1A2. In order to examine metabolic profiles of 4-n-NP in the NT, CYP1A1 and CYP1A2 cultures, the suspensions were exposed to 10 mg 1(-1) of 14C-4-n-NP using a two-liquid-phase system with carrier n-hexadecane and 192 h of incubation. Results obtained resembled those of the low concentration study. The oxidative metabolic profiles determined after hydrolytic cleavage using GC-EIMS were similar in the NT, CYP1A1 and CYP1A2 cultures. Main metabolites were side-chain mono-hydroxylated derivatives of 4-n-NP with 6'-, 7'- and 8'-OH-4-n-NP as prominent metabolites. In addition, olefinic side-chain hydroxy, ring methoxylated, keto and ring hydroxylated derivatives were observed. The lack of differences in metabolic profiles among the CYP1A1, CYP1A2 and NT cultures was referred to the low enzymatic activity of CYP1A1 and CYP1A2 as compared to the higher endogenous oxidative capacity of tobacco, as well as to similar metabolic profiles of 4-n-NP produced by CYP1A1 and CYP1A2 and tobacco itself.