Preterm neonates exposed to painful NICU procedures exhibit increased pain scores and alterations in oxygenation and heart rate. It is unclear whether these physiologic 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 experienced a TDP (n=38) 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<0.001). UA significantly decreased over time in control neonates but remained stable in TDP neonates (132.76μM to 123.23μM vs.140.50μM to 138.9μM, P=0.002). MDA levels decreased over time in control neonates but increased in TDP neonates (2.07μM to 1.81μM vs. 2.07μM to 2.21μM, P=0.01). 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.

Objective To examine the effects of sucrose on pain and biochemical markers of adenosine trisphosphate(ATP) degradation and oxidative stress in preterm neonates experiencing a clinically required heel lance. Study design Preterm neonates that met study criteria (n=131) were randomized into three groups: (1) control; (2) heel lance treated with placebo and non-nutritive sucking (NNS); and (3) heel lance treated with sucrose and NNS. Plasma markers of ATP degradation (hypoxanthine, xanthine and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the Premature Infant Pain Profile (PIPP). Data were analyzed using repeated measures ANOVA and Spearman rho. Results We found significant increases in plasma hypoxanthine and uric acid over time in neonates who received sucrose. We also found a significant negative correlation between plasma allantoin concentration and PIPP in a subgroup of neonates who received sucrose. Conclusion A single dose of oral sucrose, given before heel lance, significantly increased ATP utilization and oxidative stress in premature neonates. Because neonates are given multiple doses of sucrose per day, randomized trials are needed to examine the effect of repeated sucrose administration on ATP degradation, oxidative stress and cell injury.

IGF-II is a potent mitogen and inhibitor of apoptosis in breast cancer. Regulation of IGF-II is complex and includes inhibition by tumor suppressors, stimulation by oncogenes, and imprinting and hormonal regulation by estrogens. Resveratrol (RSV) is a phytoestrogen that displays estrogen-like agonistic and antagonistic activity. Recent studies have shown that RSV inhibits the growth of breast cancer cells and may represent a potent agent in chemopreventive therapy. Because 17beta-estradiol regulates IGF-II, we hypothesized that RSV may have a similar effect on IGF-II. The present study was designed to examine whether: 1) RSV modulates IGF-II in breast cancer cells; 2) regulation of IGF-II by RSV is dependent on the ER status; and 3) IGF-II (not IGF-I) mediates RSV effects on breast cancer cells. Treatment of MCF-7 and T47D cells with RSV (10(-6) M) caused stimulation of precursor IGF-II mRNA and protein; this effect was blocked by coincubation with 17beta-estradiol (10(-9) M). Cell growth stimulated by RSV (10(-6) M) was blocked by addition of a blocking IGF-I receptor antibody, or the antiestrogen tamoxifen (10(-7) M). In contrast, RSV treatment (10(-4) M) inhibited IGF-II secretion and cell growth in MCF-7 and T47D cells. No increase in IGF-II levels is seen in estrogen receptor (-) MCF-10 cells, even though cell growth was inhibited by RSV 10(-4) M and precursor IGF-II blocked the inhibitory effect of resveratrol. No change in IGF-I was observed with RSV treatment (10(-6) to 10(-4) M). Our study demonstrates that RSV regulates IGF-II and that IGF-II mediates RSV effect on cell survival and growth in breast cancer cells.

Background Germinal matrix intraventricular hemorrhage (IVH) is the most common type of intracranial hemorrhage observed in preterm neonates. It is a precursor of poor neurocognitive development, cerebral palsy and death. The pathophysiology is not well defined, but damage to the fragile germinal matrix vasculature may be due to free radicals generated during inflammation and as a consequence of ischemia followed by reperfusion. Assessment of the oxidative stress status in these infants is therefore important. Urinary allantoin concentration was measured in preterm neonates as a marker of oxidative stress associated with IVH. Study design Urine was collected from 44 preterm neonates at four time points between 24 and 72 hours of life (HOL) and the allantoin content was determined by gas chromatography mass spectrometry (GCMS). Records were retrospectively reviewed and the incidence and severity of IVH was categorized as follows: no IVH (n=24), mild (grade 1-2) IVH (n=13) and severe (grade 3-4) IVH (n=7). Results Neonates with severe IVH showed significantly elevated allantoin levels vs subjects with no IVH from 36 HOL (0.098±0.013µmol and 0.043±0.007µmol, respectively, p=.002). The allantoin concentration remained elevated even at 72 HOL (0.079±0.014µmol and 0.033±0.008µmol, respectively, p=.021). There were no significant differences in allantoin levels in the no IVH and mild IVH groups. IVH was diagnosed by head imaging on average at about 11th post-natal day. Conclusion Urinary allantoin levels were significantly elevated during the first 3 days of life in the neonates subsequently diagnosed with severe IVH, suggesting that oxidative stress might be a crucial factor in IVH pathogenesis. Further studies are needed to assess the usefulness of urinary allantoin in early identification of preterm infants at risk for or with severe IVH, and monitoring of the response to interventions designed to prevent or treat it.

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 1-3 . 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 2 . 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 4, 5 . Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6 . 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) [7][8][9] . 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 [10][11][12][13] . 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 7 . This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14,15 . GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16,17 . Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18 . This approach proved to be sufficiently sensitive and reproducible.

A previous observation that insulin-like growth factor II (IGF-II) inhibits the cellular uptake of a lysosomal enzyme by inhibiting binding to the IGF-II/mannose 6-phosphate receptor led to the proposal that, in a cell producing IGF-II, the routing of lysosomal enzymes might be altered. To test this hypothesis MCF-7 breast cancer cells were transfected with pRc/CMV vector only (CMV) or vector containing IGF-II complementary DNA encoding either mature (M-II) or precursor (P-II) IGF-II, and the routing of cathepsin D, a predominant lysosomal enzyme in this cell line, was examined. The concentration of IGF-II in media conditioned by P-II clones (11.2 +/- 4.3 micrograms/ml) was much higher than in media conditioned by M-II clones (1.3 +/- 1.5 micrograms/ml). Metabolic labeling experiments were performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes. Cell extracts (C) and media (M) were immuno-precipitated with a cathepsin D antiserum, and immunoprecipitates were analyzed by SDS-PAGE. The mean of the C/M ratio of cathepsin D for the seven P-II clones (1.60 +/- 0.13) was significantly lower than for the six CMV clones (3.47 +/- 0.48). Similar results were obtained when conditioned M and C were examined by immunoblotting after a 48-h incubation. The mean of the C/M ratio for the seven P-II clones (11.4 +/- 1.6) was significantly lower than for the six CMV clones (24.9 +/- 5.2). There was also a strong negative correlation between the ratio of intracellular cathepsin D to extracellular cathepsin D and relative cathepsin D synthesis (r = 0.843), consistent with increased cathepsin D production in cells overexpressing IGF-II. It is concluded that endogenous IGF-II modulates the routing of cathepsin D in MCF-7 cells.

Cathepsin D (CD) is an enzyme that promotes breast cancer. CD is stored intracellularly; however, we demonstrated that IGF-II promotes CD secretion in estrogen receptor positive (ER+ ) breast cancer cells. We also showed that resveratrol (RSV) stimulates IGF-II in ER(+) breast cancer cells. Thus, we designed this study to determine whether RSV regulates CD in MCF-7, T47D (ER+ ) breast cancer cells as well as in Hs578t (cancer)

Objective To examine the effect of neonatal morbidity on ATP breakdown in late preterm infants. Study Design Urinary hypoxanthine concentration, a marker of ATP breakdown, was measured from 82 late preterm infants on days of life (DOL) 3 to 6 using high-performance liquid chromatography. Infants were grouped according to the following diagnoses: poor nippling alone (n = 8), poor nippling plus hyperbilirubinemia (n = 21), poor nippling plus early respiratory disease (n = 26), and respiratory disease alone (n = 27). Results Neonates with respiratory disease alone had significantly higher urinary hypoxanthine over DOL 3 to 6 when compared with neonates with poor nippling (P = .020), poor nippling plus hyperbilirubinemia (P < .001), and poor nippling plus early respiratory disease (P = .017). Neonates with poor nippling who received respiratory support for 2 to 3 days had significantly higher hypoxanthine compared with infants who received respiratory support for 1 day (P = .017) or no days (P = .007). Conclusions These findings suggest that respiratory disorders significantly increase ATP degradation in late premature infants.