Prem S. Shekhawat

Neonatal Cord Blood Leptin: Its Relationship to Birth Weight, Body Mass Index, Maternal Diabetes, and Steroids

Leptin is a 16-kD protein encoded by the ob/ob (obesity) gene. In rodents it plays a role in obesity, diabetes, fertility, and neuroendocrine function. In humans serum concentrations of leptin correlate with total body fat in both adults and children. We measured cord blood leptin in 186 neonates that included 82 appropriate for gestational age (AGA), 47 large for gestational age (LGA), 20 infants of diabetic mothers, 52 preterm infants, and 15 intrauterine growth-retarded (IUGR) infants. There were 16 pairs of twins. The mothers of 17 preterm infants were treated with steroids before delivery. Leptin (mean ± SD) concentration in term, AGA infants (39.4 ± 1.1 wk) with birth weight (BW) of 3.2 ± 0.3 kg, body mass index (BMI) of 12.6 ± 1.1 was 4.01 ± 3.5 ng/mL. BW correlated with cord leptin (p = 0.002) in a multivariate analysis controlling for potential confounders. Both LGA infants and infants of diabetic mothers had higher cord leptin concentration 7.3 ± 3.8 and 6.1 ± 4.8 ng/mL, respectively, compared with AGA infants (p < 0.05). Preterm infants had a mean leptin level of 1.8 ± 0.97 ng/mL and a 3-fold elevation was seen if mothers received steroids antenatally (p = 0.006). IUGR infants had increased leptin (6.5 ± 3.9 ng/mL, p= 0.03). Concerning the twin pairs, the smaller had a higher leptin level compared with larger twin (4.1 ± 9.51 versus 2.8 ± 5.14, p = NS). Neonatal cord leptin concentrations correlate well with BW and BMI. No gender differences were found in cord blood leptin. Maternal obesity had no effect on cord leptin, whereas exogenous maternal steroids increased neonatal leptin concentrations.

Fetal fatty acid oxidation disorders, their effect on maternal health and neonatal outcome: impact of expanded newborn screening on their diagnosis and management.

Mitochondrial fatty acid oxidation disorders (FAOD) are recessively inherited errors of metabolism. Newborns with FAOD typically present with hypoketotic hypoglycemia, metabolic acidosis, hepatic failure, and cardiomyopathy. Late presentations include episodic myopathy, neuropathy, retinopathy, and arrhythmias. Sudden unexpected death can occur at any age and can be confused with sudden infant death syndrome. Some FAOD are associated with intrauterine growth restriction, prematurity, and pregnancy complications in the heterozygous mother, such as severe preeclampsia, acute fatty liver of pregnancy (AFLP), or hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. Maternal pregnancy complications occur primarily in mothers carrying a fetus with long-chain l-3-hydroxyacyl CoA dehydrogenase deficiency or general trifunctional protein deficiencies. FAOD as a group represent the most common inborn errors of metabolism, and presymptomatic diagnosis of FAOD is the key to reduce morbidity and avoid mortality. The application of tandem mass spectrometry to newborn screening provides an effective means to identify most FAOD patients presymptomatically. At the beginning of 2005, 36 state newborn screening programs have mandated or adopted this technology resulting in a marked increase in the number of asymptomatic neonates with FAOD diagnosed. To ensure the long-term benefits of such screening programs, pediatricians and other health care providers must be educated about these disorders and their treatment.

Carnitine deficiency in OCTN2-/- newborn mice leads to a severe gut and immune phenotype with widespread atrophy, apoptosis and a pro-inflammatory response.

We have investigated the gross, microscopic and molecular effects of carnitine deficiency in the neonatal gut using a mouse model with a loss-of-function mutation in the OCTN2 (SLC22A5) carnitine transporter. The tissue carnitine content of neonatal homozygous (OCTN2−/−) mouse small intestine was markedly reduced; the intestine displayed signs of stunted villous growth, early signs of inflammation, lymphocytic and macrophage infiltration and villous structure breakdown. Mitochondrial β-oxidation was active throughout the GI tract in wild type newborn mice as seen by expression of 6 key enzymes involved in β-oxidation of fatty acids and genes for these 6 enzymes were up-regulated in OCTN2−/− mice. There was increased apoptosis in gut samples from OCTN2−/− mice. OCTN2−/− mice developed a severe immune phenotype, where the thymus, spleen and lymph nodes became atrophied secondary to increased apoptosis. Carnitine deficiency led to increased expression of CD45-B220+ lymphocytes with increased production of basal and anti-CD3-stimulated pro-inflammatory cytokines in immune cells. Real-time PCR array analysis in OCTN2−/− mouse gut epithelium demonstrated down-regulation of TGF-β/BMP pathway genes. We conclude that carnitine plays a major role in neonatal OCTN2−/− mouse gut development and differentiation, and that severe carnitine deficiency leads to increased apoptosis of enterocytes, villous atrophy, inflammation and gut injury.

Carnitine Content and Expression of Mitochondrial β-Oxidation Enzymes in Placentas of Wild-Type (OCTN2 +/+ ) and OCTN2 Null (OCTN2 −/− ) Mice

Placenta requires energy to support its rapid growth, maturation, and transport function. Fatty acids are used as energy substrates in placenta, but little is known about the role played by carnitine in this process. We have investigated the role of carnitine in the expression of the enzymes involved in fatty acid β-oxidation in placenta of OCTN2−/− mice with defective carnitine transporter (OCTN2). Heterozygous (OCTN2+/−) female mice were mated with heterozygous (OCTN2+/−) male mice. Pregnant mice were killed and fetuses and placentas were collected. Carnitine was measured using HPLC and tandem mass spectrometry. Immunohistochemistry was used to detect enzyme expression. Enzyme activities were measured spectrophotometrically. The fetal and placental weights were similar among the three genotypes (OCTN2+/+, OCTN2+/−, and OCTN2−/−). The levels of carnitine were markedly reduced (<20%) in homozygous OCTN2−/− null fetuses and placentas compared with wild-type OCTN2+/+ controls. However, carnitine concentration in placenta was 2- to 7-fold higher than in the fetus in all three genotypes. Immunohistochemistry revealed that β-oxidation enzymes are expressed in trophoblast cells. Catalytic activities of these enzymes were present at comparable levels in wild-type (OCTN2+/+) and homozygous (OCTN2−/−) mouse placentas, with the exception of SCHAD, for which activity was significantly higher in OCTN2−/− placentas than in OCTN2+/+ placentas. These data show that placental OCTN2 is obligatory for accumulation of carnitine in placenta and fetus, that fatty acid β-oxidation enzymes are expressed in placenta, and that reduced carnitine levels up-regulate the expression of SCHAD in placenta.

Carnitine content and expression of mitochondrial beta-oxidation enzymes in placentas of wild-type (OCTN2(+/+)) and OCTN2 Null (OCTN2(-/-)) Mice.

Placenta requires energy to support its rapid growth, maturation, and transport function. Fatty acids are used as energy substrates in placenta, but little is known about the role played by carnitine in this process. We have investigated the role of carnitine in the expression of the enzymes involved in fatty acid β-oxidation in placenta of OCTN2−/− mice with defective carnitine transporter (OCTN2). Heterozygous (OCTN2+/−) female mice were mated with heterozygous (OCTN2+/−) male mice. Pregnant mice were killed and fetuses and placentas were collected. Carnitine was measured using HPLC and tandem mass spectrometry. Immunohistochemistry was used to detect enzyme expression. Enzyme activities were measured spectrophotometrically. The fetal and placental weights were similar among the three genotypes (OCTN2+/+, OCTN2+/−, and OCTN2−/−). The levels of carnitine were markedly reduced (<20%) in homozygous OCTN2−/− null fetuses and placentas compared with wild-type OCTN2+/+ controls. However, carnitine concentration in placenta was 2- to 7-fold higher than in the fetus in all three genotypes. Immunohistochemistry revealed that β-oxidation enzymes are expressed in trophoblast cells. Catalytic activities of these enzymes were present at comparable levels in wild-type (OCTN2+/+) and homozygous (OCTN2−/−) mouse placentas, with the exception of SCHAD, for which activity was significantly higher in OCTN2−/− placentas than in OCTN2+/+ placentas. These data show that placental OCTN2 is obligatory for accumulation of carnitine in placenta and fetus, that fatty acid β-oxidation enzymes are expressed in placenta, and that reduced carnitine levels up-regulate the expression of SCHAD in placenta.

Necrotizing Enterocolitis in a mouse model leads to widespread renal inflammation, acute kidney injury and disruption of renal tight junction proteins

Background:Necrotizing enterocolitis (NEC) is a devastating condition affecting premature infants and leads to high mortality and chronic morbidity. Severe form of NEC is associated with acute renal failure, fluid imbalance, hyponatremia, and acidosis. We investigated the effect of NEC on tight junction (TJ) proteins in kidneys using a NEC mouse model to investigate the basis for the observed renal dysfunction.Methods:NEC was induced in C57BL/6 mice by formula feeding and subjecting them to periods of hypoxia and cold stress. NEC was confirmed by gross and histological examination. We studied various markers of inflammation in kidneys and investigated changes in expression of several TJ proteins and AQP2 using immunofluorecent staining and western blotting.Results:We found markedly increased expression of NFκB, TGFβ, and ERK1/2 along with claudin-1, -2, -3, -4, -8, and AQP-2 in NEC kidneys. The membrane localization of claudin-2 was altered in the NEC kidneys and its immunostaining signal at TJ was disrupted.Conclusion:NEC led to a severe inflammatory response not only in the gut but also in the kidneys. NEC increased expression of several TJ proteins and caused disruption of claudin-2 in renal tubules. These observed changes can help explain some of the clinical findings observed in NEC.

Enzymes involved in l-carnitine biosynthesis are expressed by small intestinal enterocytes in mice: Implications for gut health

BACKGROUND Carnitine is essential for mitochondrial β-oxidation of long-chain fatty acids. Deficiency of carnitine leads to severe gut atrophy, ulceration and inflammation in animal models of carnitine deficiency. Genetic studies in large populations have linked mutations in the carnitine transporters OCTN1 and OCTN2 with Crohns disease (CD), while other studies at the same time have failed to show a similar association and report normal serum carnitine levels in CD patients. METHODS In this report, we have studied the expression of carnitine-synthesizing enzymes in intestinal epithelial cells to determine the capability of these cells to synthesize carnitine de novo. We studied expression of five enzymes involved in carnitine biosynthesis, namely 6-N-trimethyllysine dioxygenase (TMLD), 4-trimethylaminobutyraldehyde dehydrogenase (TMABADH), serine hydroxymethyltransferase 1 and 2 (SHMT1 and 2) and γ-butyrobetaine hydroxylase (BBH) by real-time PCR in mice (C3H strain). We also measured activity of γ-BBH in the intestine using an ex vivo assay and localized its expression by in situ hybridization. RESULTS Our investigations show that mouse intestinal epithelium expresses all five enzymes required for de novo carnitine biosynthesis; the expression is localized mainly in villous surface epithelial cells throughout the intestine. The final rate-limiting enzyme γ-BBH is highly active in the small intestine; its activity was 9.7 ± 3.5 pmol/mg/min, compared to 22.7 ± 7.3 pmol/mg/min in the liver. CONCLUSIONS We conclude that mouse gut epithelium is able to synthesize carnitine de novo. This capacity to synthesize carnitine in the intestine may play an important role in gut health and can help explain lack of clinical carnitine deficiency signs in subjects with mutations with OCTN transporters.

Shorter anogenital distance correlates with the severity of hypospadias in pre-pubertal boys

STUDY QUESTION Do pre-pubertal boys with hypospadias have a shorter anogenital distance (AGD) than boys with normal genitalia? SUMMARY ANSWER AGD is significantly shorter in boys with hypospadias and decreases with the severity of hypospadias. WHAT IS KNOWN ALREADY Animal studies have shown that androgen disruption and exposure to endocrine disrupting chemicals during a critical time period in early gestation, termed the male programming window (MPW), result in hypospadias and reduced AGD; and the severity of hypospadias correlates with the reduction in AGD. However, this correlation has not been established in humans. STUDY DESIGN, SIZE, DURATION A prospective descriptive study involving measurement of AGD in pre-pubertal boys (n = 458) presenting to our pediatric urology clinic with hypospadias and normal genitalia was performed over a period of 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS AGD was measured in pre-pubertal boys from 5 months to 14 years of age presenting to our clinic with hypospadias (n = 180: four were excluded) and compared with randomly selected boys with normal genitalia (controls, n = 274). Three variants of AGD, from the midpoint of the anus to base of the scrotum (AGD-AS), to the anterior base of penis (AGD-1) and to the posterior base of penis (AGD-2), were measured and assessed for correlation with the severity of hypospadias. Severity of hypospadias was classified as anterior, middle and posterior according to the meatal location. MAIN RESULTS AND THE ROLE OF CHANCE No significant difference in weight (P = 0.123), age (P = 0.162) or height (P = 0.591) between the two groups was observed. Only AGD-AS was significantly shorter in boys with hypospadias compared with controls (mean ± SD: 40.6 ± 9.7 mm versus 45.6 ± 9.4 mm, P < 0.001). This relation persisted after adjusting AGD for weight, height and age (β = 0.016, 95% confidence interval: 0.10-0.21; P < 0.001). The Spearman test showed a significant negative correlation for the severity of hypospadias with all the three AGD measures. Analysis of variance between anterior, middle and posterior subgroups showed a significant reduction in mean AGD-AS (P = 0.003) and AGD-2 (P = 0.008). LIMITATIONS, REASONS FOR CAUTION No data were collected pertaining to in utero exposure to endocrine disrupting chemicals (EDCs) or cigarette smoke, or current diet and environmental exposure to EDCs, which may have influenced the AGD. Family history of genital malformation and use of IVF were not known. There may have been a selection bias as only boys presenting to our clinic were included. WIDER IMPLICATIONS OF THE FINDINGS The findings suggest that prenatal androgens during early gestation influence development of the male reproductive system and support the existence of a MPW in humans. Of the three AGDs, AGD-AS may be the most reliable biomarker of this in utero androgen action. However, no direct link to any specific exposure leading to shortened AGD in pre-pubertal boys with hypospadias could be determined. Further large scale multi-center studies are needed to understand this association better. STUDY FUNDING/COMPETING INTERESTS Funding was from the Hypospadias Foundation. No conflicts of interest to disclose.

Prevalence of diabetes and obesity in association with prematurity and growth restriction

Intrauterine growth restriction (IUGR) is when fetuses and newborn infants have not reached their true growth potential as genetically defined. Fetuses with IUGR develop in a less than ideal environment that leads to epigenetic changes and marks infants’ metabolism for the rest of their lives. Epigenetic changes affect insulin-like growth factor-1 (IGF-1) levels and lead to insulin resistance and ultimately to a metabolic syndrome. The metabolic syndrome is a constellation of illnesses that raise one’s risk for type 2 diabetes mellitus, coronary artery disease, and ischemic heart disease, including hypertension, dyslipidemia, central obesity, insulin resistance, and inflammation. The association between IUGR or prematurity and long-term insulin resistance, obesity, hypertension, and metabolic syndrome remains unclear. While studies have shown an association, others have not supported such association. If alteration of intrauterine growth can ultimately lead to the development of metabolic derangements in childhood and adulthood, and if such association is true, then early interventions targeting the health of pregnant women will ensure the health of the population to follow.

Relationship between packed red blood cell transfusion and severe form of necrotizing enterocolitis: A case control study

ObjectiveTo determine if packed red blood cell transfusion is associated with onset of necrotizing enterocolitis, and whether withholding feed has any association with it.MethodsCase records of 100 preterm neonates, (<34 weeks gestation) who developed necrotizing enterocolitis and 99 random age-and gestation-matched controls were evaluated for any blood transfusion 48 h before onset of necrotizing enterocolitis.ResultsDuring the study period 26% infants received packed red blood cell transfusion within 48-hours prior to onset of disease and 84% of these infants were not fed around the time of transfusion. Infants who developed necrotizing enterocolitis after transfusion were older, of lower gestational age, birth weight and more likely to develop stage 3 disease. They had a lower hematocrit at birth and before onset of disease and withholding feeds around transfusion did not prevent necrotizing enterocolitis. Odds of mortality in these infants was 2.83 (95% CI 0.97-8.9) and survivors had no significant difference in incidence of periventricular leukomalacia and length of hospital stay.ConclusionBlood Transfusion associated necrotizing enterocolitis is a severe, mainly surgical form of disease.