Procedural pain and oxidative stress in premature neonates.

UNLABELLED: Preterm neonates exposed to painful procedures in the neonatal intensive care unit exhibit increased pain scores and alterations in oxygenation and heart rate. It is unclear whether these physiological responses increase the risk of oxidative stress. Using a prospective study design, we examined the relationship between a tissue-damaging procedure (TDP; tape removal during discontinuation of an indwelling central arterial or venous catheter) and oxidative stress in 80 preterm neonates. Oxidative stress was quantified by measuring uric acid (UA) and malondialdehyde (MDA) concentration in plasma before and after neonates (n = 38) experienced a TDP compared to those not experiencing any TDP (control group, n = 42). Pain was measured before and during the TDP using the Premature Infant Pain Profile (PIPP). We found that pain scores were higher in the TDP group compared to the control group (median scores, 11 and 5, respectively; P < .001). UA significantly decreased over time in control neonates but remained stable in TDP neonates (132.76 to 123.23 µM versus 140.50 to 138.9 µM; P = .002). MDA levels decreased over time in control neonates but increased in TDP neonates (2.07 to 1.81 µM versus 2.07 to 2.21 µM, P = .01). We found significant positive correlations between PIPP scores and MDA. Our data suggest a significant relationship between procedural pain and oxidative stress in preterm neonates. PERSPECTIVE: This article presents data describing a significant relationship between physiological markers of neonatal pain and oxidative stress. The method described in this paper can potentially be used to assess the direct cellular effects of procedural pain as well the effectiveness of interventions performed to decrease pain.

Biochemical measurement of neonatal hypoxia.

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA). Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase. We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin. This approach proved to be sufficiently sensitive and reproducible.

An animal model for measuring the effect of common NICU procedures on ATP metabolism.

Neonates exposed to common neonatal intensive care unit (NICU) procedures show alterations in heart rate, blood pressure, and oxygen saturation. However, it is unclear if these physiologic changes increase adenosine triphosphate (ATP) utilization, thus potentially increasing the risk for tissue hypoxia in medically fragile neonates. Plasma uric acid is a commonly used marker of increased ATP utilization because uric acid levels increase when ATP consumption is enhanced. To examine the effect of a common NICU procedure on plasma uric acid concentration, we developed a model that allows for acute monitoring of this biochemical marker in plasma in 7- to 9-day-old rabbits. In our pilot study, we exposed neonatal rabbits to a single heel lance 2.5 hr after catheter placement. We measured uric acid concentration before and 30 min after the heel lance and compared findings to levels in control animals not exposed to the heel lance. Our pilot data shows a significant difference in uric acid concentration over time between the control and heel lance groups (46.2 ± 7.1 µM vs. 54.7 ± 5.8 µM, respectively, p = .027). Calculation of percentage change from baseline showed uric acid concentration increasing in rabbits exposed to heel lance and decreasing in control rabbits (1.5 ± 4.7% vs. -16.1 ± 4.2%, respectively, p = .03). These data suggest that this animal model can be successfully used to examine the biochemical effect of common NICU procedures, such as heel lance, on markers of ATP breakdown and purine metabolism.

Necrotizing enterocolitis is associated with neonatal intestinal injury.

PURPOSE: We hypothesized that a subset of premature newborns has subclinical, intestinal mucosal compromise that predisposes to the development of necrotizing enterocolitis (NEC) days or weeks later. METHODS: Fifty-five newborns of 23 to 36 weeks' gestational age were identified, and urine was collected over the first 90 hours of life. The urinary concentration of intestinal fatty acid binding protein (iFABP(u)), a sensitive marker for intestinal injury, was determined. The diagnosis of NEC was based upon clinical condition, pathology, and/or imaging findings. RESULTS: Neonatal iFABP(u) exceeded 800 pg/mL in 27 subjects, including 9 of 9 who subsequently developed stage 2 or 3 NEC. This degree of iFABP(u) elevation, but not asphyxia, was significantly associated with the development of NEC (P < .01). CONCLUSION: In this population of premature newborns, there was a substantial incidence of intestinal mucosal compromise. All infants who subsequently developed stage 2 or 3 NEC had an elevated iFABP(u). This finding suggests a model for the pathogenesis of some cases of NEC, whereby perinatal mucosal injury predisposes to further damage when feedings are initiated. In addition, neonatal iFABP(u) assessment may represent a tool to identify infants at the highest risk for NEC and allow for the institution of focused, preventive measures.