The relationship of red blood cell transfusion to intestinal mucosal injury in premature infants.

OBJECTIVE: To determine the incidence of intestinal mucosal injury before and after transfusions in premature infants. STUDY DESIGN: Urine was collected throughout the hospital stay of 62 premature infants and specimens obtained within 24h before and after transfusion were assayed for intestinal fatty acid binding protein (iFABP). A urinary iFABP:creatinine ratio (iFABPu:Cru) of 2.0pg/nmol was considered elevated. RESULT: Forty-nine infants were transfused. iFABPu:Cru was elevated following 71 (75.6%) of 94 transfusions for which urine was available. In 51 (71.8%) of these, iFABPu:Cru was also elevated prior to the transfusion. Among four cases of transfusion-associated NEC, iFABPu was elevated following every sentinel transfusion and prior to three of them. CONCLUSION: Subclinical intestinal mucosal injury is frequent following blood transfusions in premature infants and, when present, usually precedes transfusion. This suggests that transfusion may not be a primary mediator of intestinal injury so much as anemia and its associated conditions. LEVEL OF EVIDENCE: Prognosis study/level 3.

Urinary Allantoin Is Elevated in Severe Intraventricular Hemorrhage in the Preterm Newborn.

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. 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). Neonates with severe IVH showed significantly elevated allantoin levels vs subjects with no IVH from 36 HOL (0.098 ± 0.013 µmol and 0.043 ± 0.007 µmol, respectively, p = 0.002). The allantoin concentration remained elevated even at 72 HOL (0.079 ± 0.014 µmol and 0.033 ± 0.008 µmol, respectively, p = 0.021). 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 postnatal day. 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.

Oral sucrose for heel lance enhances adenosine triphosphate use in preterm neonates with respiratory distress.

OBJECTIVE: To examine the effects of oral sucrose on procedural pain, and on biochemical markers of adenosine triphosphate utilization and oxidative stress in preterm neonates with mild to moderate respiratory distress. STUDY DESIGN: Preterm neonates with a clinically required heel lance that met study criteria (n = 49) were randomized into three groups: (1) control (n = 24), (2) heel lance treated with placebo and non-nutritive sucking (n = 15) and (3) heel lance treated with sucrose and non-nutritive sucking (n = 10). Plasma markers of adenosine triphosphate 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. Data were analyzed using repeated measures analysis of variance, chi-square and one-way analysis of variance. RESULTS: We found that in preterm neonates who were intubated and/or were receiving ⩾30% FiO2, a single dose of oral sucrose given before a heel lance significantly increased markers of adenosine triphosphate use. CONCLUSION: We found that oral sucrose enhanced adenosine triphosphate use in neonates who were intubated and/or were receiving ⩾30% FiO2. Although oral sucrose decreased pain scores, our data suggest that it also increased energy use as evidenced by increased plasma markers of adenosine triphosphate utilization. These effects of sucrose, specifically the fructose component, on adenosine triphosphate metabolism warrant further investigation.

Early detection of impending necrotizing enterocolitis with urinary intestinal fatty acid-binding protein.

BACKGROUND: Necrotizing enterocolitis (NEC) is diagnosed after the development of feeding intolerance and characteristic physical and imaging findings. Earlier detection of a subclinical prodrome might allow for the institution of measures that could prevent or attenuate the severity of the disease. OBJECTIVES: We sought to determine whether urinary intestinal fatty acid-binding protein (iFABPu) might be elevated prior to the first clinical manifestations of NEC. METHODS: Urine was collected daily from 62 infants of a gestational age of 24-28 weeks. Based on clinical, imaging and operative findings, subjects were determined to have Bell stage 2 or 3 NEC. In all the subjects with NEC and in 21 age-matched controls, iFABPu was determined using an ELISA, and was expressed in terms of its ratio to urinary creatinine (Cr), i.e. iFABPu/Cru. Receiver operating characteristic (ROC) curves were constructed to define the predictive value of iFABPu/Cru for impending NEC in the days prior to the first clinical manifestations. RESULTS: Five subjects developed NEC (stage 2: n = 3 and stage 3: n = 2). The day before the first clinical manifestation of NEC, a ROC curve showed that an iFABPu/Cru >10.2 pg/nmol predicted impending NEC with a sensitivity of 100% and a specificity of 95.6%. iFABPu/Cru did not predict NEC 2 days prior to the first sign of disease. CONCLUSIONS: An elevated iFABPu was a sensitive and specific predictor of impending NEC 1 day prior to the first clinical manifestations. iFABPu screening might identify infants at a high risk and allow for the institution of measures that could ameliorate or prevent NEC.

Urinary Hypoxanthine as a Measure of Increased ATP Utilization in Late Preterm Infants.

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.

Oral sucrose for heel lance increases adenosine triphosphate use and oxidative stress in preterm neonates.

OBJECTIVE: To examine the effects of sucrose on pain and biochemical markers of adenosine triphosphate (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 3 groups: (1) control; (2) heel lance treated with placebo and non-nutritive sucking; and (3) heel lance treated with sucrose and non-nutritive sucking. 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 with the Premature Infant Pain Profile. Data were analyzed by the use of 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 pain scores and plasma allantoin concentration in a subgroup of neonates who received sucrose. CONCLUSION: A single dose of oral sucrose, given before heel lance, significantly increased ATP use and oxidative stress in premature neonates. Because neonates are given multiple doses of sucrose per day, randomized trials are needed to examine the effects of repeated sucrose administration on ATP degradation, oxidative stress, and cell injury.

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.

Insulin-like growth factor II mediates resveratrol stimulatory effect on cathepsin D in breast cancer 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) and MCF-10A (normal) ER - cell lines. RSV (10(- 6) M) increased CD and IGF-II secretion in ER+ but not ER - cells. RSV treatment (10(- 4) M) inhibited CD in ER+ but not in ER - cells. Transfection of ER - cells with proIGF-II increased CD secretion. RSV (10(- 6) M) modulates CD secretion through IGF-II while RSV (10(- 4) M) inhibits CD in ER+ but not ER - cells. Regulation of CD by RSV represents a novel mechanism by which RSV may protect against breast cancer.

Resveratrol regulates insulin-like growth factor-II in breast cancer cells.

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.