Lower oxygen consumption and Complex I activity in mitochondria isolated from skeletal muscle of fetal sheep with intrauterine growth restriction.

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower hindlimb oxygen consumption rates (OCRs), indicating depressed mitochondrial oxidative phosphorylation capacity in their skeletal muscle. We hypothesized that OCRs are lower in skeletal muscle mitochondria from IUGR fetuses, due to reduced electron transport chain (ETC) activity and lower abundances of tricarboxylic acid (TCA) cycle enzymes. IUGR sheep fetuses (n = 12) were created with mid-gestation maternal hyperthermia and compared with control fetuses (n = 12). At 132 ± 1 days of gestation, biceps femoris muscles were collected, and the mitochondria were isolated. Mitochondria from IUGR muscle have 47% lower State 3 (Complex I-dependent) OCRs than controls, whereas State 4 (proton leak) OCRs were not different between groups. Furthermore, Complex I, but not Complex II or IV, enzymatic activity was lower in IUGR fetuses compared with controls. Proteomic analysis (n = 6/group) identified 160 differentially expressed proteins between groups, with 107 upregulated and 53 downregulated mitochondria proteins in IUGR fetuses compared with controls. Although no differences were identified in ETC subunit protein abundances, abundances of key TCA cycle enzymes [isocitrate dehydrogenase (NAD+) 3 noncatalytic subunit ß (IDH3B), succinate-CoA ligase ADP-forming subunit-ß (SUCLA2), and oxoglutarate dehydrogenase (OGDH)] were lower in IUGR mitochondria. IUGR mitochondria had a greater abundance of a hypoxia-inducible protein, NADH dehydrogenase 1α subcomplex 4-like 2, which is known to incorporate into Complex I and lower Complex I-mediated NADH oxidation. Our findings show that mitochondria from IUGR skeletal muscle adapt to hypoxemia and hypoglycemia by lowering Complex I activity and TCA cycle enzyme concentrations, which together, act to lower OCR and NADH production/oxidation in IUGR skeletal muscle.

[The activity of enzymes of amino acid metabolism of the placenta in different terms of the physiological and complicated pregnancy.]

The activity of amino acid metabolism enzymes and the content of free amino acids in the placenta during physiological pregnancy and placental insufficiency (PI) were studied using spectrophotometric methods and ion-exchange chromatography. It was found that in PI placental activity of the studied enzymes: alanine-, cysteine-e, tyrosine-, glutamino- transferase, glutathione synthetase, glutamate dehydrogenase decreases at different periods of gestation. The opposite variations occur for aspartataminotranferase and glutaminase. Similar changes are detected for amino acids synthesized or used in the course of appropriate reactions: aspartic acid, glutamic acid, glutamine, alanine, cysteine, tyrosine, arginine. The correlation between enzyme activity and amino acid content was revealed. Different periods of pregnancy are characterized by varying degrees of change, especially expressed in the second trimester, characterized by the most intense growth and development of the fetus, and its increased needs for trophic material. The revealed changes obviously play a pathogenetic role in the formation and further development of PI.

[Features of redox processes in the amniotic fluid at placental insufficiency]. / Osobennosti okislitel'no-vosstanovitel'nykh protsessov v okoloplodnykh vodakh pri platsentarnoi nedostatochnosti.

Activity of prooxidant enzymes (NADPH-oxidase and xanthine oxidase), antioxidant enzymes (superoxide dismutase (SOD) and catalase), enzymes of the glutathione-dependent systems, as well as antioxidant vitamins (retinol and a-tocopherol), lipid peroxidation products (LPP) (conjugated dienes and Schiff bases), and peroxide chemiluminescence were studied in the amniotic fluid at different periods of physiological pregnancy and placental insufficiency (PI). It was found that at PI the activity of NADPH-oxidase, xanthine oxidase increased and the activity of SOD, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase and the content of fat-soluble vitamins decreased. The direct and inverse correlation between the studied pro- and antioxidant parameters and the content of LPP products, was found ro be different in the II and III trimesters of gestation. The revealed differences obviously reflect metabolic impairments in the fetoplacental complex, and the activity and level of the paremeters of redox processes can be used as tests for pre- and postnatal disorders.

AMPK and Placental Progenitor Cells.

AMPK is important in numerous physiological systems but plays a vital role in embryonic and placental development. The placenta is a unique organ that is the essential lifeline between the mother and baby during pregnancy and gestation. During placental development, oxygen concentrations are very low until cells differentiate to establish the appropriate lineages that take on new functions required for placental and embryonic survival. Balancing the oxygen regulatory environment with the demands for energy and need to maintain metabolism during this process places AMPK at the center of maintaining placental cellular homeostasis as it integrates and responds to numerous complex stimuli. AMPK plays a critical role in sensing metabolic and energy changes. Once activated, it turns on pathways that produce energy and shuts down catabolic processes. AMPK coordinates cell growth, differentiation, and nutrient transport to maintain cell survival. Appropriate regulation of AMPK is essential for normal placental and embryonic development, and its dysregulation may lead to pregnancy-associated disorders such as intrauterine growth restriction, placental insufficiency, or preeclampsia.

[Proteolytic activity of fetoplacental complex in norm and pathology].

The activity of angiotensin converting enzyme (ACE), carboxypeptidase N (CPN), and leucine aminopeptidase (LAP) has been investigated in the fetoplacental complex (FPC) in normal and placental insufficiency (FPI). ACE and LAP activities were significantly higher in the placental tissue than in maternal serum and umbilical vein serum. CPN activity was significantly lower in umbilical vein serum as compared to that of women in childbirth. Probably, the studied enzymes are involved in formation of reduced sensitivity of FPC of blood vessels during physiological pregnancy. In cases of placental insufficiency a significant increase of LAP activity was found in the placental tissue and umbilical vein serum. In addition, the pathological course of pregnancy caused a significant increase of CPN activity in serum of pregnant women in comparison to the norm. The obtained data suggest that during FPI proteolytic enzymes participate in the formation of compensatoty-adaptive reactions in the FPC. Results of this study are interesting in context of development of methods for prevention and correction of metabolic disorders in pathologies of pregnancy.

The long and short of it: the role of telomeres in fetal origins of adult disease.

Placental insufficiency, maternal malnutrition, and other causes of intrauterine growth restriction (IUGR) can significantly affect short-term growth and long-term health. Following IUGR, there is an increased risk for cardiovascular disease and Type 2 Diabetes. The etiology of these diseases is beginning to be elucidated, and premature aging or cellular senescence through increased oxidative stress and DNA damage to telomeric ends may be initiators of these disease processes. This paper will explore the areas where telomere and telomerase biology can have significant effects on various tissues in the body in IUGR outcomes.

Effect of age and gender on the progression of adult vascular dysfunction in a mouse model of fetal programming lacking endothelial nitric oxide synthase.

The objective of this study was to investigate vascular function at different ages in a transgenic murine model of fetal vascular programming using a model of uteroplacental insufficiency induced by lack of endothelial nitric oxide synthase. Homozygous NOS3 knockout (KO) and wild-type (WT) mice were cross bred to produce WT, KO, and heterozygous that developed in WT (KOP) or KO (KOM) mothers. Male/female offspring from the four groups were killed at 7, 14, and 21 wk of age (n = 5-10/group), and carotid arteries were used for in vitro vascular studies. Responses to phenylephrine (PE), with/without N(G)-nitro-L-arginine methyl ester (L-NAME), angiotensin (ANG), acetylcholine (ACh), sodium nitroprusside, and isoproterenol (ISO) were studied. At 7 wk, only KO offspring showed higher contractile response to PE, whereas, at 14 and 21 wk, both KO and KOM had a higher response. Incubation with L-NAME abolished these differences. ANG contraction was higher in male KO in all age groups and in 21-wk-old females. Relaxation to ACh and ISO was absent in KO, and significantly decreased in KOM offspring in all age groups compared with KOP and WT, independent of gender. Sodium nitroprusside was not different between groups. The effect of the altered intrauterine environment on the development of abnormal vascular function was limited at 7 wk of age and most evident at 14 wk; further deterioration was limited to ANG-mediated vascular contractility in KO offspring. Our findings provide some hope that at least the first seven postnatal weeks may be an appropriate therapeutic window to prevent cardiovascular disease later in life.

[Detection of serum fucosidase activity].

A procedure has been developed for simultaneous determination of the activity of a-L-fucosidase (EC 3.2.1.5 1) and the beta-D-fucosidase activity caused by beta-D-galactosidase (EC 3.2.1.23, Form IV) in serum. The paper presents data on changes in the above serum enzymatic activities in an experiment, in children with recurrent laryngotracheitis and in pregnant females with gestosis and placental insufficiency.

Uteroplacental insufficiency affects epigenetic determinants of chromatin structure in brains of neonatal and juvenile IUGR rats.

Intrauterine growth retardation (IUGR) increases the risk of neuroendocrine reprogramming. In the rat, IUGR leads to persistent changes in cerebral mRNA levels. This suggests lasting alterations in IUGR cerebral transcriptional regulation, which may result from changes in chromatin structure. Candidate nutritional triggers for these changes include altered cerebral zinc and one-carbon metabolite levels. We hypothesized that IUGR affects cerebral chromatin structure in neonatal and postnatal rat brains. Rats were rendered IUGR by bilateral uterine artery ligation; controls (Con) underwent sham surgery. At day of life 0 (d0), we measured cerebral DNA methylation, histone acetylation, expression of chromatin-affecting enzymes, and cerebral levels of one-carbon metabolites and zinc. At day of life 21 (d21), we measured cerebral DNA methylation and histone acetylation, as well as the caloric content of Con and IUGR rat breast milk. At d0, IUGR significantly decreased genome-wide and CpG island methylation, as well as increased histone 3 lysine 9 (H3/K9) and histone 3 lysine 14 (H3/K14) acetylation in the hippocampus and periventricular white matter, respectively. IUGR also decreased expression of the chromatin-affecting enzymes DNA methyltransferase 1 (DNMT1), methyl-CpG binding protein 2 (MeCP2), and histone deacetylase (HDAC)1 in association with increased cerebral levels of zinc. In d21 female IUGR rats, cerebral CpG DNA methylation remained lower, whereas H3/K9 and H3/K14 hyperacetylation persisted in hippocampus and white matter, respectively. In d21 male rats, IUGR decreased acetylation of H3/K9 and H3/K14 in these respective regions compared with controls. Despite these differences, caloric, fat, and protein content were similar in breast milk from Con and IUGR dams. We conclude that IUGR results in postnatal changes in cerebral chromatin structure and that these changes are sex specific.

Investigation of the role of epigenetic modification of the rat glucokinase gene in fetal programming.

Fetal malnutrition is associated with development of impaired glucose tolerance, diabetes and hypertension in later life in humans and several mammalian species. The mechanisms that underlie this phenomenon of fetal programming are unknown. We hypothesize that adverse effects in utero and early life may influence the basal expression levels of certain genes such that they are re-set with long-term consequences for the organism. An excellent candidate mechanism for this re-setting process is DNA methylation, since post-natal methylation patterns are largely established in utero. We have sought to test this hypothesis by investigating the glucokinase gene (Gck) in rat offspring programmed using a maternal low protein diet model (MLP). Northern blot reveals that fasting levels of Gck expression are reduced after programming, although this distinction disappears after feeding. Bisulphite sequencing of the hepatic Gck promoter indicates a complete absence of methylation at the 12 CpG sites studied in controls and MLP animals. Non-expressing cardiac tissue also showed no DNA methylation in this region, whereas brain and all fetal tissues were fully methylated. These findings are not consistent with the hypothesis that programming results from differential methylation of Gck. However, it remains possible that programming may influence methylation patterns in Gck at a distance from the promoter, or in genes encoding factors that regulate basal Gck expression.

Production of NO and oxidative destruction of proteins in the placenta during normal pregnancy and placental insufficiency.

Study of NO generation and oxidative destruction of proteins showed intensification of these processes in the placenta during normal gestation. Activity of NO synthase and the concentration of nitrites in the placenta in women with placental insufficiency and spontaneous abortions were below the normal. Full-term pregnancy against the background of placental insufficiency was associated with a compensatory increase in NO production in the placenta. On the other hand, oxidative destruction of proteins increased in placental insufficiency irrespective of pregnancy outcome.

Cotyledon and binucleate cell nitric oxide synthase expression in an ovine model of fetal growth restriction.

Heat exposure early in ovine pregnancy results in placental insufficiency and intrauterine growth restriction (PI-IUGR). We hypothesized that heat exposure in this model disrupts placental structure and reduces placental endothelial nitric oxide synthase (eNOS) protein expression. We measured eNOS protein content and performed immunohistochemistry for eNOS in placentas from thermoneutral (TN) and hyperthermic (HT) animals killed at midgestation (90 days). Placental histomorphometry was compared between groups. Compared with the TN controls, the HT group showed reduced delivery weights (457 +/- 49 vs. 631 +/- 21 g; P < 0.05) and a trend for reduced placentome weights (288 +/- 61 vs. 554 +/- 122 g; P = 0.09). Cotyledon eNOS protein content was reduced by 50% in the HT group (P < 0.03). eNOS localized similarly to the vascular endothelium and binucleated cells (BNCs) within the trophoblast of both experimental groups. HT cotyledons showed a reduction in the ratio of fetal to maternal stromal tissue (1.36 +/- 0.36 vs. 3.59 +/- 1.2; P< or = 0.03). We conclude that eNOS protein expression is reduced in this model of PI-IUGR and that eNOS localizes to both vascular endothelium and the BNC. We speculate that disruption of normal vascular development and BNC eNOS production and function leads to abnormal placental vascular tone and blood flow in this model of PI-IUGR.

Placenta of discordant twins: lack of change in histochemically detectable enzyme activities.

We localised three important enzymes histochemically in placental trophoblasts from women who gave birth to dichorionic discordant twins, in which the co-twin was affected by foetal growth restriction (FGR). The enzymes studied were adenosine diphosphate-degrading enzyme (ADP-degrading enzyme, plasma membrane enzyme), cytochrome c oxidase (mitochondrial enzyme), and glucose-6-phosphatase (endoplasmic reticular enzyme). We compared these enzyme activities and their distribution patterns among placentas of the smaller (FGR) co-twin, larger co-twin, pre-eclamptic singleton with FGR, and normal singletons with birth weight of appropriate for their gestational ages. In FGR co-twin placentas, the intensity and localisation pattern of these three enzymes did not differ from those seen in the larger co-twin and normal singleton placentas. Decreased ADP-degrading activity and cytochrome c oxidase negative mitochondria, which were characteristic features of pre-eclamptic trophoblasts, were not observed in FGR co-twin placentas. These observations indicated that, in the FGR co-twin, enzyme-histochemically detectable trophoblastic cell dysfunction may be absent, or if present, less prominent, compared with pre-eclamptic FGR. We previously reported that placental trophoblasts from singleton idiopathic FGR also showed no reduction in these enzyme activities. In mechanism and pathophysiology, FGR in dichorionic discordant twins may be quite different from pre-eclamptic FGR, but somewhat resembles idiopathic FGR, though all three disorders lead to placental insufficiency, resulting in limited foetal growth.

[N-acetyl-beta-glucosaminidase (NAG) activity in parturients with placental insufficiency in postmature pregnancy and with EPH-gestosis]. / Aktywnosc N-acetylo-beta-glukozoaminidazy (NAG) u rodzacych z niewydolnoscia lozyska w ciazy przenoszonej i z EPH-gestoza.

NAG activity evaluation was carried out in parturients' blood and placental homogenates in regular pregnancies (n = 46) and complicated with biological postmaturity (n = 30) and EPH-gestosis (n = 24). It was revealed that in the blood of parturients with pregnancy complicated with gestosis there was considerable increase of NAG activity (2.43 +/- 1.02 microKat/kg). Lower activity level was found in parturients with entopic pregnancy (1.93 +/- 0.87 microKat/kg), and the lowest in those with postmature pregnancy (1.78 +/- 0.56 microKat/kg). The placental homogenates presented statistically significant differences--the highest in postmature pregnancy (6.37 +/- 2.01 mKat/kg), lower in pregnancy complicated with gestosis (4.85 +/- 1.52 mKat/kg) and the lowest in normal pregnancy (3.52 +/- 1.21 mKat/kg). The elevated activity in blood serum of parturient with gestosis may indicate kidney damage in the course of disease. High activity in homogenate may indicate the processes of placental degradation in postmature pregnancy. There is no evidence of correlation between NAG activity in blood and activity in placental tissue.

Serum heat-stable alkaline phosphatase activity in normal pregnancy.

OBJECTIVE: To determine the reference values for heat-stable alkaline phosphatase in apparently healthy non-pregnant and in normal pregnant females and to assess the suitability of this parameter as a measure of placental function and indirectly as a measure of foetal survival. DESIGN: A prospective study. SETTING: Ante-natal clinic, University of Nigeria Teaching Hospital, Enugu, Nigeria. PARTICIPANTS: Ninety eight apparently normal pregnant females in various gestational ages and 47 apparently healthy non-pregnant females. MAIN OUTCOME MEASURES: The serum heat-stable alkaline phosphatase activity. RESULTS: Variability in values exists between individuals in the same gestational age. The mean and standard deviation of serum heat-stable alkaline phosphatase was found to be 0.79 +/- 1.54 iu/L in the control group. No significant difference was found (p < 0.05) between this and the mean and standard deviation of serum alkaline phosphatase of 0.98 +/- 1.28 iu/L found in the first eight weeks of gestation. However the means and standard deviations of first trimester, second and third trimester were significantly higher than the controls. CONCLUSION: In the face of worsening economic situation in third world countries this simple method could be used in urban and some rural hospitals in assessing placenta insufficiency.

Vascular placental insufficiency in the rabbit. Changes in creatine kinase and adenylate kinase activities in fetal tissues.

Vascular placental insufficiency is considered a common pathogenic factor in human intrauterine growth retardation. To mimic this condition, the rabbit, a 'perinatal brain developer' was utilized as an experimental model. Ischemic conditions were achieved by total ligation of approximately 30% of the uteroplacental vessels of half of the fetuses in each pregnant rabbit in the last third of gestation. The change in activity of the brain type isozyme of creatine kinase (CKBB), involved in energy regeneration and regulation, was assessed as a response marker to tissue ischemia in rabbit tissues: cerebellum, cerebrum, kidney, liver and placenta. A significant transient increase in CK-specific activity was found in the kidney and the cerebellum but not in the other organs tested, at 24 and 48 h after ligation. This increase was not seen with adenylate kinase, another enzyme involved in energy regeneration and regulation. It is proposed that an increase in CK-specific activity could serve as a metabolic marker of vascular insufficiency in rapidly developing tissues, representing part of a compensatory mechanism to overcome an energetic gap induced by ischemia.

Cord blood serum creatine kinase isoenzymes with placental dysfunction.

It has been suggested that perinatal asphyxia is not generally followed by neurological impairment unless there is preexisting chronic fetal distress. In cases of brain damage one can observe elevated levels of CK-BB. The purpose of our study was to evaluate CK isoenzymes in umbilical cord blood sera of newborns affected by chronic fetal distress. Fetal distress reflected by placental dysfunction was characterized by a diminished HPL level and decreased activity of CAP. We estimated CK isoenzymes with the use of DEAE-sepharose CL-6B column chromatography. Total CK activity was measured using kits supplied by Boehringer-Mannheim (Monotest CK-NAC aktiviert). The clinical state of examined newborns was estimated. Investigations were carried out in the group of 57 infants delivered after 37 weeks of gestation. Total CK activity in cord sera ranged from 40 to 400 U/l. Our results showed a significant rise of CK-BB activity in cord sera of newborns delivered from pregnancies with placental dysfunction (figure 2) as well as in cases of asphyxiated infants (figure 3). We were unable to demonstrate differences in total CK, CK-MM and CK-MB activities in all examined groups of newborns. Other authors have confirmed that severe asphyxia results in increase in CK-BB activity in cord blood. Infants with ominous fetal heart rate patterns have higher CK-BB activity. There are several possible sources for CK-BB activity in umbilical cord blood sera, i.e. fetal brain, lung, gastrointestinal tract, placenta and uterus. It appears that the brain is most likely the source of elevated CK-BB activity found in cord blood in cases of placental dysfunction.

Alkaline ribonuclease activity in maternal serum and in serum of newborns from birth up to thirty days of age. A study made with full-term appropriate-, full-term small- and preterm appropriate-for-gestational-age infants.

We have studied the alkaline ribonuclease (RNase) activity in maternal serum and serum of full-term small- (T-SGA), full-term appropriate- (T-AGA) and preterm appropriate-for-gestational age (PT-AGA) newborns. A significantly lower level of RNase was observed in T-AGA and T-SGA newborns on the 30th day of age and in PT-AGA newborns on the 15th and 30th days of age, as compared to other T-AGA, T-SGA and PT-AGA groups of infants at birth. RNase activity was significantly higher in cord blood than in the maternal blood in all categories studied. Moreover, in preterm newborns, RNase activity in cord blood was significantly higher in those presenting a lower gestational age. We did not observe any significant difference in RNase levels in the cord blood of newborns from the 3 categories studied. The same results were observed concerning maternal blood. We, therefore, conclude that RNase activity in cord blood or in maternal blood is not a very satisfactory indicator of fetal malnutrition.