- Email: [email protected]
P34
Abstracts / Nitric Oxide 31 (2013) S13–S48
NO-dependent vasodilation using MAHMA NONOate was sensitive to PDE5 inhibition at both 21% and 1% O2.These data suggest a hypoxia-dependent shift from a PDE5-sensitive to PDE5-insensitive mechanism for nitrite-dependent vasodilation. Conclusion: Collectively, these data indicate the possible role for 8nitro-cGMP in nitrite-mediated hypoxic signaling paradigms. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.034
P33 Nitrite prevents hypoxic injury of neurons through suppression of caspase-6-mediated cleavage of Tau Yen-Jung Chen a,b, Tzu-Ching Meng a,b a National Taiwan University, Taipei, Taiwan b Academia Sinica, Taipei, Taiwan It has been shown clearly that hypoxic stress associated with various pathological conditions, such as ischemic stroke or obstructive sleep apnea, impairs signal transduction of neurons, thus causing severe destruction of brain functions. To date the underlying mechanism of hypoxia-induced neuronal injuries remains elusive. It is also not known what strategy might be used to prevent neurons from hypoxic damages. Here we show that exposure of differentiated Neuro-2a (N2a) cells and primary rat cortical neurons with hypoxia resulted in significant neurite/axon retraction, a hallmark of neuronal injury. Interestingly, this detrimental effect of hypoxia on neurites was completely prevented by nitrite treatment, suggesting a protective role of nitrite in neuronal functions. We further illustrated that hypoxic stress induces collapse and disassembly of microtubule structure, the main framework composing neurites, leading to retraction of neurites. The disruption of microtubule in neurites is caused at least in part by the loss of integrity of Tau, a microtubule-associated protein that stabilizes microtubule structure, through a caspase-dependent process. Our data showed that cleavage of Tau and retraction of neurites is induced by caspase-6, which is robustly activated in hypoxic neurons. Importantly, in the presence of nitrite, caspase-6 was inactivated through S-nitrosylation of its active-site Cys residue, thus being unable to catalyze the proteolysis of Tau. Together these results are the first to demonstrate that nitrite plays a critical role in the protection of neuronal function against hypoxic insult. Our findings show that nitrite holds great potential for the treatment of diseases associated with acute or chronic hypoxia-induced disorder of neuronal homeostasis. Disclosure: Nothing to disclose.
S27
including DNA synthesis and microtubule formation. Recent data suggests that nitrite may play a novel role in reducing mitogen-stimulated cell proliferation. While nitrite has been shown to reduce cell proliferation during both normoxia and hypoxia, the mechanism remains to be elucidated. Myoglobin is known to have several interactions with nitrite and other reactive nitrogen species, including nitric oxide (NO) scavenging during normoxia and nitrite reduction to form bioavailable NO during hypoxia. Most recently, myoglobin has been found to be abnormally expressed in several malignant epithelial tissues including breast, lung, colon, and ovarian tumors. Limited data suggests that myoglobin expression results in decreased tumor volumes, although the mechanism for this effect remains unclear. Hypothesis: In the present study, we hypothesize that myoglobin expression regulates the ability of nitrite to modulate cell proliferation in malignancy. Methods: We used physiological modulators, such as hypoxia and lactate, to regulate myoglobin levels in the breast cancer cell lines MCF7 and MB-MDA-231. Additionally, rat aortic smooth muscle cells (RASMC), which have abundant myoglobin expression, were used to determine the effect of myoglobin on regulating nitrite-mediated inhibition of cell proliferation. Results: We demonstrate using RASMCs, containing abundant levels of myoglobin, that nitrite promotes mitochondrial fusion and the inhibition of mitogen-stimulated cell proliferation during normoxia by increasing two mitochondrial fusion signals: mitofusin 1 (Mfn-1) expression and the inhibitory phosphorylation of dynamin-related protein 1 (DRP1). Increased Mfn-1 levels are correlated with increased p21Waf1/Cip1 expression, and nitrite treatment does not increase p21Waf1/Cip1 expression in the presence of Mfn-1 siRNA, suggesting a direct effect of nitrite-mediated Mfn-1 expression on subsequent p21Waf1/Cip1 expression and cell cycle regulation. We observe in breast cancer cell lines that myoglobin expression can be induced during either prolonged hypoxia, or during normoxia through the addition of lactate to culture media. These physiological inductions of myoglobin are accompanied by inhibition of cell proliferation, an effect that is further attenuated by the addition of nitrite. Further, in breast cancer cells during hypoxia, upregulation of myoglobin and reduced cell proliferation is correlated with increased Mfn-1 and p21Waf1/Cip1 expression. Upon addition of lactate to normoxic breast cancer cells, we observed p21Waf1/Cip1 induction along with increased myoglobin expression, decreased mitochondrial respiration, and decreased cell proliferation. Conclusions: Taken together, these data implicate the regulation of mitochondrial dynamics in nitrite-mediated decreases in cell proliferation. Further, these data also suggest that myoglobin expression may play a critical role in modulating this pathway. Further under-
Nitrite blood levels 20
Background: Rapid and uncontrolled cell proliferation is a hallmark of malignancy. In many tumor types, uncontrolled cell growth results in tumor metastasis and poor patient outcome. Many pathways involved in cell proliferation are targeted by standard chemotherapy
5
Time in minutes Fig. 1.
T6 0 R
T4 9 R
T1 5 R
R
T4
0
lin e
Kelly Quesnelle, Li Mo, Yinna Wang, Sergey Zharikov, Catherine Corey, Donna Beer Stolz, Brian Zuckerbraun, Sruti Shiva University of Pittsburgh, Pittsburgh, PA
Drug A (8umol) Drug C (2umol) Placebo
10
as e
Myoglobin and nitrite regulate cell proliferation via changes in mitochondrial dynamics
15
B
P34
Nitrite level in uM
http://dx.doi.org/10.1016/j.niox.2013.02.035
S28
Abstracts / Nitric Oxide 31 (2013) S13–S48
standing and elucidation of this pathway is warranted, as nitrite may be a novel anti-cancer agent in tumors harboring aberrant myoglobin expression. Disclosure: Nothing to disclose.
due to tissue saturation with higher doses, or in the half-life. These results are guiding present studies of nitrite in more lethal VF. Disclosure: Supported by research grants from the Max Kade Foundation Inc. and the Laerdal Foundation for Acute Medicine.
http://dx.doi.org/10.1016/j.niox.2013.02.036 http://dx.doi.org/10.1016/j.niox.2013.02.037
P35 P36 Nitrite as a novel resuscitation adjunct after ventricular fibrillation cardiac arrest in rats: A preliminary report Thomas Uray a, Tomas Drabek a, Jason P. Stezoski a, Keri Janesko-Feldman b, Samuel A. Tisherman a, Patrick M. Kochanek a, Cameron Dezfulian a a University of Pittsburgh School of Medicine, Safar Center for Resusciation Research, Department of Critical Care Medicine, Pittsburgh, PA, United States b Safar Center for Resusciation Research, Pittsburgh, PA, United States Background: Beside therapeutic hypothermia no novel therapies have been developed to improve outcomes of patients suffering car diac arrest (CA). In rat models, nitrite NO2 has the theoretical advantage of being converted locally to nitric oxide (NO) in ischemic regions improving microcirculation. Nitrite reduced neurological damage after asphyxial CA in rats. Whether nitrite is protective in CA due to ventricular fibrillation (VF) is unclear. Therefore, we developed a novel VF rat CA model to test this hypothesis and characterize nitrite’s pharmacokinetics in this disease model. Methods: In this pilot randomized blinded trial, adult male Sprague–Dawley rats were anesthetized, intubated and catheterized (arterial and venous). VF was induced using a bipolar pacing catheter and left untreated for 5 min. Manual cardiopulmonary resuscitation (CPR) with drugs (epinephrine/sodium bicarbonate) and defibrillation was initiated using a predefined protocol simulating current practice. After return of spontaneous circulation (ROSC), rats were randomized to 500 lL placebo (Plasmalyte), low-dose (2 lM) or high-dose (8 lM) intravenous nitrite given as a 5 min infusion at 5 min and 50 min after CPR-start. Nitrite whole blood levels were measured at baseline, before and after each of 2 drug infusions. Hemodynamics and temperature were measured over 60 min after CPR-start; temperature was controlled (37 °C) for 12 h post-arrest. Neurologic Deficit Score (NDS) was assessed on post-arrest days 1– 2 and fear-conditioning (a hippocampal-mediated behavior) on days 7–8. On day 8, rats were perfused with formalin and surviving hippocampal CA1 neurons were quantified using H&E and FluoroJade-B staining. Statistics are presented as median and interquartile range between 25th and 75th percentiles. Results: Nine of 10 animals randomized into the study after ROSC survived until day 8. CPR-duration was 52 (49–56) s. At 15 min after initiation of CPR, blood nitrite levels (see Fig. 1) were 4.7 (4.7– 4.7) lM in the 8 lM and 2.1 (1.9–2.4) lM in the 2 lM groups vs. 0.8 (0.7–0.9) lM in placebo (p < 0.01). At 60 min, nitrite levels were 15.8 (15.6–15.9) lM and 3.5 (3.1–3.9) lM in the 8 lM and 2 lM groups, respectively, vs. 0.8 (0.7–1.0) lM in placebo (p < 0.001; Fig. 1) There were no significant differences between the treatment groups and placebo in regards to baseline nitrite levels, hemodynamics, temperature, NDS, fear conditioning, or CA1 neuronal survival, which was approximately 99 (51–133) cells/mm. Conclusion: In this feasibility study, nitrite can be safely administered early after ROSC in a novel though mild model of VF CA. The clearance of the first nitrite dose was faster than the second dose suggesting either alteration in the volume of distribution, perhaps
Nitrite and nitrate concentrations and metabolism in breast milk, artificial milk, and parenteral nutrition of term and preterm infants Jesica Jones a, Janet R. Ninnis a, Andrew O. Hopper a, Yomna Ibrahim a, T. Allen Merritt a, Kim-Wah Wan b, Gordon G. Power c, Arlin B. Blood a,c a Loma Linda University, Department of Pediatrics, Division of Neonatology, Loma Linda, CA, United States b Loma Linda University, School of Pharmacy, Loma Linda, CA, United States c Loma Linda University, Center for Perinatal Biology, Loma Linda, CA, United States Background: In the adult, dietary nitrate is converted to nitrite by bacteria in the mouth. In the acidic environment of the stomach, the ingested nitrite is converted to NO where it is has a protective effect by increasing blood flow and mucus production, and by modulating the microbial flora of the GI tract. Ingested nitrite has also been linked to improved cardiovascular function in adults. However, the intake and importance of nitrate and nitrite in newborn infants is largely unknown. The purpose of this work is to determine nitrite and nitrate concentrations in both the diet and total parenteral nutrition (TPN) of term and preterm infants, and to characterize the means by which nitrite is metabolized in fresh and freeze– thawed breast milk. Methods: We measured nitrate and nitrite levels in breast milk collected from mothers of preterm and term infants, ten different brands of commonly used infant formula, and TPN. We also measured the effect of freeze–thawing, temperature and oxygen tension variation, and several metal-containing enzyme antagonists on the metabolism of nitrite in samples of breast milk. Results: Nitrite concentrations averaged 0.07 ± 0.01 lM in the milk of mothers of preterm infants, significantly less than in the milk of mothers of term infants (0.13 ± 0.02 lM) (P < 0.01). Nitrate concentrations averaged 13.6 ± 3.7 lM in the milk of mothers of preterm infants and 12.7 ± 4.9 lM in the milk of mothers of term infants. Freeze–thawing resulted in a 65 ± 6% decrease in nitrite concentrations. Nitrite disappeared from breast milk with a half-life of 32 min at 37 °C, and slowed at lower temperatures (Q10 = 1.5). The metabolism was dependent upon oxygen and rapidly accelerated by its increase. The metabolic rate was not affected by de-lipidation of the milk, the addition of N-ethylmaleimide to block thiol groups, or the addition of allopurinol to block xanthine oxidase. The metabolism of nitrite was inhibited by the addition of ferricyanide. Nitrite metabolism was also inhibited through the use of three different blockers of lactoperoxidase, a normal constituent of breast milk, and was increased by the addition of purified lactoperoxidase, suggesting that this enzyme is involved in nitrite metabolism. Nitrite concentrations in infant formula varied widely and ranged from 0.01 to 1.3 lM, and nitrate concentrations ranged from 12 to 67 lM. The nitrite (0.08 ± 0.02 lM) and nitrate (9.5 ± 0.8 lM) concentrations in TPN were similar to those measured in fresh breast milk.
NO-dependent vasodilation using MAHMA NONOate was sensitive to PDE5 inhibition at both 21% and 1% O2.These data suggest a hypoxia-dependent shift from a PDE5-sensitive to PDE5-insensitive mechanism for nitrite-dependent vasodilation. Conclusion: Collectively, these data indicate the possible role for 8nitro-cGMP in nitrite-mediated hypoxic signaling paradigms. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.034
P33 Nitrite prevents hypoxic injury of neurons through suppression of caspase-6-mediated cleavage of Tau Yen-Jung Chen a,b, Tzu-Ching Meng a,b a National Taiwan University, Taipei, Taiwan b Academia Sinica, Taipei, Taiwan It has been shown clearly that hypoxic stress associated with various pathological conditions, such as ischemic stroke or obstructive sleep apnea, impairs signal transduction of neurons, thus causing severe destruction of brain functions. To date the underlying mechanism of hypoxia-induced neuronal injuries remains elusive. It is also not known what strategy might be used to prevent neurons from hypoxic damages. Here we show that exposure of differentiated Neuro-2a (N2a) cells and primary rat cortical neurons with hypoxia resulted in significant neurite/axon retraction, a hallmark of neuronal injury. Interestingly, this detrimental effect of hypoxia on neurites was completely prevented by nitrite treatment, suggesting a protective role of nitrite in neuronal functions. We further illustrated that hypoxic stress induces collapse and disassembly of microtubule structure, the main framework composing neurites, leading to retraction of neurites. The disruption of microtubule in neurites is caused at least in part by the loss of integrity of Tau, a microtubule-associated protein that stabilizes microtubule structure, through a caspase-dependent process. Our data showed that cleavage of Tau and retraction of neurites is induced by caspase-6, which is robustly activated in hypoxic neurons. Importantly, in the presence of nitrite, caspase-6 was inactivated through S-nitrosylation of its active-site Cys residue, thus being unable to catalyze the proteolysis of Tau. Together these results are the first to demonstrate that nitrite plays a critical role in the protection of neuronal function against hypoxic insult. Our findings show that nitrite holds great potential for the treatment of diseases associated with acute or chronic hypoxia-induced disorder of neuronal homeostasis. Disclosure: Nothing to disclose.
S27
including DNA synthesis and microtubule formation. Recent data suggests that nitrite may play a novel role in reducing mitogen-stimulated cell proliferation. While nitrite has been shown to reduce cell proliferation during both normoxia and hypoxia, the mechanism remains to be elucidated. Myoglobin is known to have several interactions with nitrite and other reactive nitrogen species, including nitric oxide (NO) scavenging during normoxia and nitrite reduction to form bioavailable NO during hypoxia. Most recently, myoglobin has been found to be abnormally expressed in several malignant epithelial tissues including breast, lung, colon, and ovarian tumors. Limited data suggests that myoglobin expression results in decreased tumor volumes, although the mechanism for this effect remains unclear. Hypothesis: In the present study, we hypothesize that myoglobin expression regulates the ability of nitrite to modulate cell proliferation in malignancy. Methods: We used physiological modulators, such as hypoxia and lactate, to regulate myoglobin levels in the breast cancer cell lines MCF7 and MB-MDA-231. Additionally, rat aortic smooth muscle cells (RASMC), which have abundant myoglobin expression, were used to determine the effect of myoglobin on regulating nitrite-mediated inhibition of cell proliferation. Results: We demonstrate using RASMCs, containing abundant levels of myoglobin, that nitrite promotes mitochondrial fusion and the inhibition of mitogen-stimulated cell proliferation during normoxia by increasing two mitochondrial fusion signals: mitofusin 1 (Mfn-1) expression and the inhibitory phosphorylation of dynamin-related protein 1 (DRP1). Increased Mfn-1 levels are correlated with increased p21Waf1/Cip1 expression, and nitrite treatment does not increase p21Waf1/Cip1 expression in the presence of Mfn-1 siRNA, suggesting a direct effect of nitrite-mediated Mfn-1 expression on subsequent p21Waf1/Cip1 expression and cell cycle regulation. We observe in breast cancer cell lines that myoglobin expression can be induced during either prolonged hypoxia, or during normoxia through the addition of lactate to culture media. These physiological inductions of myoglobin are accompanied by inhibition of cell proliferation, an effect that is further attenuated by the addition of nitrite. Further, in breast cancer cells during hypoxia, upregulation of myoglobin and reduced cell proliferation is correlated with increased Mfn-1 and p21Waf1/Cip1 expression. Upon addition of lactate to normoxic breast cancer cells, we observed p21Waf1/Cip1 induction along with increased myoglobin expression, decreased mitochondrial respiration, and decreased cell proliferation. Conclusions: Taken together, these data implicate the regulation of mitochondrial dynamics in nitrite-mediated decreases in cell proliferation. Further, these data also suggest that myoglobin expression may play a critical role in modulating this pathway. Further under-
Nitrite blood levels 20
Background: Rapid and uncontrolled cell proliferation is a hallmark of malignancy. In many tumor types, uncontrolled cell growth results in tumor metastasis and poor patient outcome. Many pathways involved in cell proliferation are targeted by standard chemotherapy
5
Time in minutes Fig. 1.
T6 0 R
T4 9 R
T1 5 R
R
T4
0
lin e
Kelly Quesnelle, Li Mo, Yinna Wang, Sergey Zharikov, Catherine Corey, Donna Beer Stolz, Brian Zuckerbraun, Sruti Shiva University of Pittsburgh, Pittsburgh, PA
Drug A (8umol) Drug C (2umol) Placebo
10
as e
Myoglobin and nitrite regulate cell proliferation via changes in mitochondrial dynamics
15
B
P34
Nitrite level in uM
http://dx.doi.org/10.1016/j.niox.2013.02.035
S28
Abstracts / Nitric Oxide 31 (2013) S13–S48
standing and elucidation of this pathway is warranted, as nitrite may be a novel anti-cancer agent in tumors harboring aberrant myoglobin expression. Disclosure: Nothing to disclose.
due to tissue saturation with higher doses, or in the half-life. These results are guiding present studies of nitrite in more lethal VF. Disclosure: Supported by research grants from the Max Kade Foundation Inc. and the Laerdal Foundation for Acute Medicine.
http://dx.doi.org/10.1016/j.niox.2013.02.036 http://dx.doi.org/10.1016/j.niox.2013.02.037
P35 P36 Nitrite as a novel resuscitation adjunct after ventricular fibrillation cardiac arrest in rats: A preliminary report Thomas Uray a, Tomas Drabek a, Jason P. Stezoski a, Keri Janesko-Feldman b, Samuel A. Tisherman a, Patrick M. Kochanek a, Cameron Dezfulian a a University of Pittsburgh School of Medicine, Safar Center for Resusciation Research, Department of Critical Care Medicine, Pittsburgh, PA, United States b Safar Center for Resusciation Research, Pittsburgh, PA, United States Background: Beside therapeutic hypothermia no novel therapies have been developed to improve outcomes of patients suffering car diac arrest (CA). In rat models, nitrite NO2 has the theoretical advantage of being converted locally to nitric oxide (NO) in ischemic regions improving microcirculation. Nitrite reduced neurological damage after asphyxial CA in rats. Whether nitrite is protective in CA due to ventricular fibrillation (VF) is unclear. Therefore, we developed a novel VF rat CA model to test this hypothesis and characterize nitrite’s pharmacokinetics in this disease model. Methods: In this pilot randomized blinded trial, adult male Sprague–Dawley rats were anesthetized, intubated and catheterized (arterial and venous). VF was induced using a bipolar pacing catheter and left untreated for 5 min. Manual cardiopulmonary resuscitation (CPR) with drugs (epinephrine/sodium bicarbonate) and defibrillation was initiated using a predefined protocol simulating current practice. After return of spontaneous circulation (ROSC), rats were randomized to 500 lL placebo (Plasmalyte), low-dose (2 lM) or high-dose (8 lM) intravenous nitrite given as a 5 min infusion at 5 min and 50 min after CPR-start. Nitrite whole blood levels were measured at baseline, before and after each of 2 drug infusions. Hemodynamics and temperature were measured over 60 min after CPR-start; temperature was controlled (37 °C) for 12 h post-arrest. Neurologic Deficit Score (NDS) was assessed on post-arrest days 1– 2 and fear-conditioning (a hippocampal-mediated behavior) on days 7–8. On day 8, rats were perfused with formalin and surviving hippocampal CA1 neurons were quantified using H&E and FluoroJade-B staining. Statistics are presented as median and interquartile range between 25th and 75th percentiles. Results: Nine of 10 animals randomized into the study after ROSC survived until day 8. CPR-duration was 52 (49–56) s. At 15 min after initiation of CPR, blood nitrite levels (see Fig. 1) were 4.7 (4.7– 4.7) lM in the 8 lM and 2.1 (1.9–2.4) lM in the 2 lM groups vs. 0.8 (0.7–0.9) lM in placebo (p < 0.01). At 60 min, nitrite levels were 15.8 (15.6–15.9) lM and 3.5 (3.1–3.9) lM in the 8 lM and 2 lM groups, respectively, vs. 0.8 (0.7–1.0) lM in placebo (p < 0.001; Fig. 1) There were no significant differences between the treatment groups and placebo in regards to baseline nitrite levels, hemodynamics, temperature, NDS, fear conditioning, or CA1 neuronal survival, which was approximately 99 (51–133) cells/mm. Conclusion: In this feasibility study, nitrite can be safely administered early after ROSC in a novel though mild model of VF CA. The clearance of the first nitrite dose was faster than the second dose suggesting either alteration in the volume of distribution, perhaps
Nitrite and nitrate concentrations and metabolism in breast milk, artificial milk, and parenteral nutrition of term and preterm infants Jesica Jones a, Janet R. Ninnis a, Andrew O. Hopper a, Yomna Ibrahim a, T. Allen Merritt a, Kim-Wah Wan b, Gordon G. Power c, Arlin B. Blood a,c a Loma Linda University, Department of Pediatrics, Division of Neonatology, Loma Linda, CA, United States b Loma Linda University, School of Pharmacy, Loma Linda, CA, United States c Loma Linda University, Center for Perinatal Biology, Loma Linda, CA, United States Background: In the adult, dietary nitrate is converted to nitrite by bacteria in the mouth. In the acidic environment of the stomach, the ingested nitrite is converted to NO where it is has a protective effect by increasing blood flow and mucus production, and by modulating the microbial flora of the GI tract. Ingested nitrite has also been linked to improved cardiovascular function in adults. However, the intake and importance of nitrate and nitrite in newborn infants is largely unknown. The purpose of this work is to determine nitrite and nitrate concentrations in both the diet and total parenteral nutrition (TPN) of term and preterm infants, and to characterize the means by which nitrite is metabolized in fresh and freeze– thawed breast milk. Methods: We measured nitrate and nitrite levels in breast milk collected from mothers of preterm and term infants, ten different brands of commonly used infant formula, and TPN. We also measured the effect of freeze–thawing, temperature and oxygen tension variation, and several metal-containing enzyme antagonists on the metabolism of nitrite in samples of breast milk. Results: Nitrite concentrations averaged 0.07 ± 0.01 lM in the milk of mothers of preterm infants, significantly less than in the milk of mothers of term infants (0.13 ± 0.02 lM) (P < 0.01). Nitrate concentrations averaged 13.6 ± 3.7 lM in the milk of mothers of preterm infants and 12.7 ± 4.9 lM in the milk of mothers of term infants. Freeze–thawing resulted in a 65 ± 6% decrease in nitrite concentrations. Nitrite disappeared from breast milk with a half-life of 32 min at 37 °C, and slowed at lower temperatures (Q10 = 1.5). The metabolism was dependent upon oxygen and rapidly accelerated by its increase. The metabolic rate was not affected by de-lipidation of the milk, the addition of N-ethylmaleimide to block thiol groups, or the addition of allopurinol to block xanthine oxidase. The metabolism of nitrite was inhibited by the addition of ferricyanide. Nitrite metabolism was also inhibited through the use of three different blockers of lactoperoxidase, a normal constituent of breast milk, and was increased by the addition of purified lactoperoxidase, suggesting that this enzyme is involved in nitrite metabolism. Nitrite concentrations in infant formula varied widely and ranged from 0.01 to 1.3 lM, and nitrate concentrations ranged from 12 to 67 lM. The nitrite (0.08 ± 0.02 lM) and nitrate (9.5 ± 0.8 lM) concentrations in TPN were similar to those measured in fresh breast milk.