Lipoprotein subfraction patterns throughout gestation in The Gambia: changes in subfraction composition and their relationships with infant birth weights.

BACKGROUND: Lipoprotein subfraction concentrations have been shown to change as gestation progresses in resource-rich settings. The objective of the current study was to evaluate the impact of pregnancy on different-sized lipoprotein particle concentrations and compositions in a resource-poor setting. METHOD: Samples were collected from pregnant women in rural Gambia at enrollment (8-20 weeks), 20 weeks, and 30 weeks of gestation. Concentrations of different-sized high-density, low-density, and triglyceride-rich lipoprotein particles (HDL, LDL, and TRL, respectively) were measured by nuclear magnetic resonance in 126 pooled plasma samples from a subset of women. HDL was isolated and the HDL proteome evaluated using mass spectroscopy. Subfraction concentrations from women in The Gambia were also compared to concentrations in women in the U.S. in mid gestation. RESULTS: Total lipoprotein particles and all-sized TRL, LDL, and HDL particle concentrations increased during gestation, with the exception of medium-sized LDL and HDL particles which decreased. Subfraction concentrations were not associated with infant birth weights, though relationships were found between some lipoprotein subfraction concentrations in women with normal versus low birth weight infants (< 2500 kg). HDL's proteome also changed during gestation, showing enrichment in proteins associated with metal ion binding, hemostasis, lipid metabolism, protease inhibitors, proteolysis, and complement activation. Compared to women in the U.S., Gambian women had lower large- and small-sized LDL and HDL concentrations, but similar medium-sized LDL and HDL concentrations. CONCLUSIONS: Most lipoprotein subfraction concentrations increase throughout pregnancy in Gambian women and are lower in Gambian vs U.S. women, the exception being medium-sized LDL and HDL particle concentrations which decrease during gestation and are similar in both cohorts of women. The proteomes of HDL also change in ways to support gestation. These changes warrant further study to determine how a lack of change or different changes could impact negative pregnancy outcomes.

Pregnancy is accompanied by larger high density lipoprotein particles and compositionally distinct subspecies.

Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother.

Maternal plasma lipid levels across pregnancy and the risks of small-for-gestational age and low birth weight: a cohort study from rural Gambia.

BACKGROUND: Sub-optimal maternal lipid levels during pregnancy may be implicated in the pathophysiological mechanisms leading to low birth weight (LBW) and small-for-gestational-age (SGA). We aimed to determine whether maternal lipid levels across pregnancy were associated with birth weight and the risks of LBW and SGA in rural Gambia. METHODS: This secondary analysis of the ENID trial involved 573 pregnant women with term deliveries. Plasma levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and triglycerides (TG) were analyzed at enrolment (mean (SD) = 13.9 (3.3) weeks gestation), 20 and 30 weeks gestation as continuous variables and percentile groups. Regression models with adjustment for confounders were used to examine associations between gestational lipid levels and birth weight and the risks of LBW (birth weight < 2500 g) and SGA (<10th percentile INTERGROWTH-21ST for birth weight). RESULTS: There were 7.9% LBW and 32.5% SGA infants. At enrolment, every unit increase in HDL-c was associated with a 2.7% (P = 0.011) reduction in relative risk of LBW. At 20 weeks gestation, every unit increase in TC levels was associated with a 1.3% reduction in relative risk of LBW (P = 0.002). Low (<10th percentile) HDL-c at enrolment or at 20 weeks gestation was associated with a 2.6 (P = 0.007) and 3.0 (P = 0.003) times greater risk of LBW, respectively, compared with referent (10th─90th) HDL-c. High (>90th percentile) LDL-c at 30 weeks gestation was associated with a 55% lower risk of SGA compared with referent LDL-c (P = 0.017). Increased levels of TC (ß = 1.3, P = 0.027) at 20 weeks gestation and of TC (ß = 1.2, P = 0.006) and LDL-c (ß = 1.5, P = 0.002) at 30 weeks gestation were all associated with higher birth weight. CONCLUSIONS: In rural Gambia, lipid levels during pregnancy were associated with infant birth weight and the risks of LBW and SGA. Associations varied by lipid class and changed across pregnancy, indicating an adaptive process by which maternal lipids may influence fetal growth and birth outcomes. TRIAL REGISTRATION: This trial was registered as ISRCTN49285450 on: 12/11/2009.

Studies in genetically modified mice implicate maternal HDL as a mediator of fetal growth.

Studies in humans have shown a direct association between maternal plasma cholesterol concentrations and infant birthweight. Similarly, previous studies in our laboratory have shown that chow-fed mice lacking apolipoprotein (apo) A-I, the major protein in HDL, have low HDL-cholesterol (HDL-C) concentrations and smaller fetuses in midgestation. In the current study, we measured fetal weights in mice with varying levels of apoA-I gene dose (knockout, wild-type, and transgenic) and examined metabolic pathways known to affect fetal growth. As expected, we found the differences in apoA-I expression led to changes in HDL particle size and protein cargo as well as plasma cholesterol concentrations. Fetal masses correlated directly with maternal plasma cholesterol and apoA-I concentrations, but placental masses and histology did not differ between groups of mice. There was no significant difference in glucose or amino acid transport to the fetus or in expression levels of the glucose (glucose transporter 1 and 2) or amino acid (sodium-coupled neutral amino acid transporter 1 and 2) transporters in whole placentas, although there was a trend for greater uptake of both nutrients in the whole fetal unit (fetus + placenta) of mice with greater apoA-I levels; significant differences in transport rates occurred when mice without apoA-I (knockout) vs. mice with apoA-I (wild-type and transgenic) were compared. Glucose tolerance tests were improved in the mice with the highest level of apoA-I, suggesting increased insulin-induced uptake of glucose by tissues of apoA-I transgenic mice. Thus, maternal HDL is associated with fetal growth, an effect that is likely mediated by plasma cholesterol or other HDL-cargo, including apolipoproteins or complement system proteins. A direct role of enhanced glucose and/or amino acid transport cannot be excluded.-Rebholz, S. L., Melchior, J. T., Davidson, W. S., Jones, H. N., Welge, J. A., Prentice, A. M., Moore, S. E., Woollett, L. A. Studies in genetically modified mice implicate maternal HDL as a mediator of fetal growth.

Apolipoprotein A-IV exerts its anorectic action through a PI3K/Akt signaling pathway in the hypothalamus.

Apolipoprotein A-IV (apoA-IV) is a satiation factor that acts in the hypothalamus, however, the intracellular mechanisms responsible for this action are still largely unknown. Here we report that apoA-IV treatment elicited a rapid activation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway in cultured primary hypothalamic neurons, and this effect was significantly attenuated by pretreatment with LY294002, an inhibitor of the PI3K pathway. To determine if the activation of PI3K is required for apoA-IV's inhibitory effect on food intake, apoA-IV was administered intracerebroventricularly. We found that apoA-IV significantly reduced food intake and activated PI3K signaling in the hypothalamus, and these effects were abolished by icv pre-treatment with LY294002. To identify the distinct brain sites where apoA-IV exerts its anorectic action, apoA-IV was administered into the ventromedial hypothalamus (VMH) through implanted bilateral cannula. At a low dose (0.5 µg), apoA-IV significantly inhibited food intake and activated PI3K signaling pathway in the VMH of lean rats, but not in high-fat diet-induced obese (DIO) rats. These results collectively demonstrate a critical role of the PI3K/Akt pathway in apoA-IV's anorectic action in lean rats and suggest a defective PI3K pathway in the VMH is responsible for the impaired apoA-IV's anorectic action in the DIO animals.

Effects of a defective endoplasmic reticulum-associated degradation pathway on the stress response, virulence, and antifungal drug susceptibility of the mold pathogen Aspergillus fumigatus.

Proteins that are destined for release outside the eukaryotic cell, insertion into the plasma membrane, or delivery to intracellular organelles are processed and folded in the endoplasmic reticulum (ER). An imbalance between the level of nascent proteins entering the ER and the organelle's ability to manage that load results in the accumulation of unfolded proteins. Terminally unfolded proteins are disposed of by ER-associated degradation (ERAD), a pathway that transports the aberrant proteins across the ER membrane into the cytosol for proteasomal degradation. The ERAD pathway was targeted in the mold pathogen Aspergillus fumigatus by deleting the hrdA gene, encoding the A. fumigatus ortholog of Hrd1, the E3 ubiquitin ligase previously shown to contribute to ERAD in other species. Loss of HrdA was associated with impaired degradation of a folding-defective ERAD substrate, CPY*, as well as activation of the unfolded-protein response (UPR). The ΔhrdA mutant showed resistance to voriconazole and reduced thermotolerance but was otherwise unaffected by a variety of environmental stressors. A double-deletion mutant deficient in both HrdA and another component of the same ERAD complex, DerA, was defective in secretion and showed hypersensitivity to ER, thermal, and cell wall stress. However, the ΔhrdA ΔderA mutant remained virulent in mouse and insect infection models. These data demonstrate that HrdA and DerA support complementary ERAD functions that promote survival under conditions of ER stress but are dispensable for virulence in the host environment.

Effect of dietary sphingomyelin on absorption and fractional synthetic rate of cholesterol and serum lipid profile in humans.

BACKGROUND: Diets enriched with sphingolipids may improve blood lipid profiles. Studies in animals have shown reductions in cholesterol absorption and alterations in blood lipids after treatment with sphingomyelin (SM). However, minimal information exists on effect of SM on cholesterol absorption and metabolism in humans. The objective was to assess the effect of SM consumption on serum lipid concentrations and cholesterol metabolism in healthy humans. METHODS: Ten healthy adult males and females completed a randomized crossover study. Subjects consumed controlled diets with or without 1 g/day SM for 14 days separated by at least 4 week washout period. Serum lipid profile and markers of cholesterol metabolism including cholesterol absorption and synthesis were analyzed. RESULTS: Serum triglycerides, total, LDL- and VLDL- cholesterol were not affected while HDL cholesterol concentrations were increased (p = 0.043) by SM diet consumption. No change in cholesterol absorption and cholesterol fractional synthesis rate was observed with supplementation of SM compared to control. Intraluminal cholesterol solubilization was also not affected by consumption of SM enriched diet. CONCLUSIONS: In humans, 1 g/day of dietary SM does not alter the blood lipid profile except for an increased HDL-cholesterol concentration and has no effect on cholesterol absorption, synthesis and intraluminal solubilization compared to control. TRIAL REGISTRATION: Clinicaltrials.gov # NCT00328211.

Multiparity leads to obesity and inflammation in mothers and obesity in male offspring.

Multiparity is an independent risk factor for obesity in parous females. In addition to being a health issue for the mother, offspring of multiparous females may also be at risk for obesity later in life. The aim of the current study was to establish a mouse model that mimics the human pathology of multiparity and determine the effects of multiparity-induced obesity (MIO) on offspring in adulthood. C57BL/6 mice were mated and studied when primiparous (1st pregnancy) or multiparous (4th pregnancy). Dams became obese with multiparity, an effect that was independent of the age of the dam. Multiparous dams also had increased markers of inflammation (JNK activation, cytokine expression) in adipose tissue and liver that was greater than inflammation in nulliparous females made obese with a high-fat diet. Placental inflammation was prevalent in multiparous vs. primiparous dams as well. Male offspring of the multiparous dams developed increased adiposity by 24 wk of age relative to the progeny of primiparous dams, although food consumption was similar in both groups. Lipid metabolism was altered in liver and fat in that mRNA levels of regulatory genes (PGC-1α) as well as metabolic genes (CPT I) and Akt phosphorylation were decreased in offspring of multiparous dams. Thus, in mice, as in humans, multiparity increases adiposity and is associated with hepatic and placental inflammation and abnormal glucose tolerance. Importantly, MIO leads to increased body fat and metabolic dysfunction in the offspring, suggesting a role in the propagation of obesity.

Reverse cholesterol transport is elevated in carboxyl ester lipase-knockout mice.

Mechanisms to increase reverse cholesterol transport (RCT) and biliary sterol disposal are currently sought to prevent atherosclerosis. Previous work with HepG2 cells and primary hepatocytes showed that carboxyl ester lipase (CEL), a broad-spectrum lipase secreted by pancreas and liver, plays an important role in hydrolysis of high-density lipoprotein (HDL) cholesteryl esters (CEs) after selective uptake by hepatocytes. The effect of CEL on RCT of HDL cholesterol was assessed by measuring biliary and fecal disposal of radiolabeled HDL-CE in control and Cel(-/-) mice. Radiolabeled CE was increased 3-fold in hepatic bile of Cel(-/-) mice, and the mass of CE in gall bladder bile was elevated. Total radiolabeled transport from plasma to hepatic bile was more rapid in Cel(-/-) mice. Fecal disposal of radiolabel from HDL-CE, as well as total sterol mass, was markedly elevated for Cel(-/-) mice, primarily due to more CE. RCT of macrophage CE was also increased in Cel(-/-) mice, as measured by excretion of radiolabel from injected J774 cells. Increased sterol loss was compensated by increased cholesterol synthesis in Cel(-/-) mice. Together, the data demonstrate significantly increased RCT in the absence of CEL and suggest a novel mechanism by which to manipulate plasma cholesterol flux.

Roles of maternal HDL during pregnancy.

BACKGROUND: High density lipoproteins (HDL) were first linked to cardiovascular disease (CVD) over 30 years ago when an inverse relationship was shown between CVD and HDL-cholesterol levels. It is now apparent that HDL composition and function, not cholesterol levels, are the pertinent measurements describing HDL's role in various disease processes, especially those with subclinical or overt inflammation. SCOPE OF REVIEW: Pregnancy is also an inflammatory state. When inflammation becomes excessive during pregnancy, there is an increased risk for adverse outcomes that affect the health of the mother and fetus, including preterm birth and preeclampsia. Though studies on HDL during pregnancy are limited, recent evidence demonstrates that HDL composition and function change during pregnancy and in women with adverse outcomes. GENERAL SIGNIFICANCE: In this review, we will discuss how HDL may play a role in maintaining a healthy pregnancy and how impairments in function could lead to pregnancies with adverse outcomes.

Dietary fat impacts fetal growth and metabolism: uptake of chylomicron remnant core lipids by the placenta.

The fetus requires significant energy for growth and development. Although glucose is a major source of energy for the fetus, other maternal nutrients also appear to promote growth. Thus, the goal of these studies was to determine whether triglyceride-rich remnants are taken up by the placenta and whether maternal dietary lipids, independently of adiposity, can impact fetal growth. To accomplish our first goal, chylomicron particles were duallly labeled with cholesteryl ester and triglycerides. The placenta took up remnant particles/core lipids at rates greater than adipose tissue and skeletal muscle but less than the liver. Although the placenta expresses apoE receptors, uptake of chylomicron remnants and/or core lipids can occur independently of apoE. To determine the impact of dietary lipid on fetal growth, independent of maternal adiposity, females were fed high-fat diets (HFD) for 1 mo; there was no change in adiposity or leptin levels prior to or during pregnancy of dams fed HFD. Fetal masses were greater in dams fed HFD, and mRNA levels of proteins involved in fatty acid oxidation (CPT I, PPARα), but not glucose oxidation (pyruvate kinase) or other regulatory processes (HNF-4α, LXR), were increased with maternal dietary fat. There was also no change in mRNA levels of proteins involved in placental glucose and fatty acid transport, and GLUT1 protein levels in microvillous membranes were similar in placentas of dams fed either diet. Thus, the ability of the placenta to take up chylomicron remnant core lipids likely contributes to accelerated fetal growth in females fed high fat diets.

Longitudinal changes in HDL-cholesterol concentration are associated with different risk factors in primiparous and nulliparous young women: The NHLBI Growth and Health Study (NGHS).

BACKGROUND: Previous studies report that first pregnancy is associated with persistent decreases in HDL-cholesterol (HDL-C) concentrations. OBJECTIVE: This study evaluated factors associated with declines in HDL-C concentration in parous and nulliparous young women. METHODS: This study leverages data from African-American and white women from the NHLBI Growth and Health Study. Parity-related changes in lipids, BMI and percent body fat were assessed longitudinally. A subset of primiparous and nulliparous women with paired lipid measurements were analyzed regarding changes in HDL-C concentrations. RESULTS: Among 870 women in longitudinal analyses, African-American women had higher parity (p<0.0001), with baseline measurements of each parity group being similar. HDL-C concentration decreased significantly and remained lower after the first pregnancy, while BMI and percent body fat increased with increasing parity. In the subset of 401 women, HDL-C concentration decreased among primiparous women (-4.81 ± 0.93 mg/dl), with no overall change in nulliparous (p = 0.003). In both groups, greater HDL-C concentration declines were independently associated with higher initial HDL-C concentration and greater increases in BMI (both p<0.0001). Among primiparous women, younger delivery age (p = 0.0001) and birth control use (p = 0.004) were associated with greater HDL-C concentration decline. Nulliparous white women's HDL-C concentration increased over time, with no change in African-American women (p = 0.008); no racial difference was seen in primiparous women. CONCLUSION: Persistent decreases in HDL-C concentration were associated with the first pregnancy, and were greater with higher initial HDL-C concentration. Racial differences in HDL-C concentration emerged over time in nulliparous women, but not primiparous women. Potential impacts of these findings on women's long-term cardiometabolic health should be evaluated.

Intestinal adaptation after ileal interposition surgery increases bile acid recycling and protects against obesity-related comorbidities.

Surgical interposition of distal ileum into the proximal jejunum is a bariatric procedure that improves the metabolic syndrome. Changes in intestinal and hepatic physiology after ileal interposition (transposition) surgery (IIS) are not well understood. Our aim was to elucidate the adaptation of the interposed ileum, which we hypothesized, would lead to early bile acid reabsorption in the interposed ileum, thus short circuiting enterohepatic bile acid recycling to more proximal bowel segments. Rats with diet-induced obesity were randomized to IIS, with 10 cm of ileum repositioned distal to the duodenum, or sham surgery. A subgroup of sham rats was pair-fed to IIS rats. Physiological parameters were measured until 6 wk postsurgery. IIS rats ate less and lost more weight for the first 2 wk postsurgery. At study completion, body weights were not different, but IIS rats had reversed components of the metabolic syndrome. The interposed ileal segment adapted to a more jejunum-like villi length, mucosal surface area, and GATA4/ILBP mRNA. The interposed segment retained capacity for bile acid reabsorption and anorectic hormone secretion with the presence of ASBT and glucagon-like-peptide-1-positive cells in the villi. IIS rats had reduced primary bile acid levels in the proximal intestinal tract and higher primary bile acid levels in the serum, suggesting an early and efficient reabsorption of primary bile acids. IIS rats also had increased taurine and glycine-conjugated serum bile acids and reduced fecal bile acid loss. There was decreased hepatic Cyp27A1 mRNA with no changes in hepatic FXR, SHP, or NTCP expression. IIS protects against the metabolic syndrome through short-circuiting enterohepatic bile acid recycling. There is early reabsorption of primary bile acids despite selective "jejunization" of the interposed ileal segment. Changes in serum bile acids or bile acid enterohepatic recycling may mediate the metabolic benefits seen after bariatric surgery.

High-fat diet before and during pregnancy causes marked up-regulation of placental nutrient transport and fetal overgrowth in C57/BL6 mice.

Maternal overweight and obesity in pregnancy often result in fetal overgrowth, which increases the risk for the baby to develop metabolic syndrome later in life. However, the mechanisms underlying fetal overgrowth are not established. We developed a mouse model and hypothesized that a maternal high-fat (HF) diet causes up-regulation of placental nutrient transport, resulting in fetal overgrowth. C57BL/6J female mice were fed a control (11% energy from fat) or HF (32% energy from fat) diet for 8 wk before mating and throughout gestation and were studied at embryonic day 18.5. The HF diet increased maternal adiposity, as assessed by fat pad weight, and circulating maternal leptin, decreased serum adiponectin concentrations, and caused a marked increase in fetal growth (+43%). The HF diet also increased transplacental transport of glucose (5-fold) and neutral amino acids (10-fold) in vivo. In microvillous plasma membranes (MVMs) isolated from placentas of HF-fed animals, protein expression of glucose transporter 1 (GLUT1) was increased 5-fold, and protein expression of sodium-coupled neutral amino acid transporter (SNAT) 2 was elevated 9-fold. In contrast, MVM protein expression of GLUT 3 or SNAT4 was unaltered. These data suggest that up-regulation of specific placental nutrient transporter isoforms constitute a mechanism linking maternal high-fat diet and obesity to fetal overgrowth.

Maternal plasma cholesterol concentration and preterm birth: a meta-analysis and systematic review of literature.

Background: Women that previously had preterm labor are at an increased risk for heart disease. Because spontaneous preterm birth is an adverse pregnancy outcome that affects millions of children worldwide, our objective was to review and analyze studies that have examined associations between maternal total cholesterol (TC), LDL-C, and HDL-C concentrations during pregnancy and the risk of preterm birth to potentially define biomarkers or targets for treatment.Method: A search was performed and 22 articles were found that examined the association of maternal plasma cholesterol concentrations and preterm birth. A meta-analysis was performed on 10 of the articles, those that used maternal lipid concentrations as the outcome and presented results as means plus variables, and a qualitative review was performed on all 22 articles.Results: The meta-analysis showed no relationship between maternal TC, LDL-C, or HDL-C and increased risk of preterm birth, although, a near significant relationship between low maternal HDL-C concentration and preterm birth (p = .055). Importantly, associations increased when cholesterol concentrations were combined with inflammatory markers or metabolic syndrome factors.Conclusions: The relationship between maternal cholesterol levels and preterm birth is heterogeneous. Associations are strengthened when maternal cholesterol concentrations are combined with other factors that may be related to more recently defined lipoprotein functions.

Hepatic bile acid metabolism in the neonatal hamster: expansion of the bile acid pool parallels increased Cyp7a1 expression levels.

Intraluminal concentrations of bile acids are low in newborn infants and increase rapidly after birth, at least partly owing to increased bile acid synthesis rates. The expansion of the bile acid pool is critical since bile acids are required to stimulate bile flow and absorb lipids, a major component of newborn diets. The purpose of the present studies was to determine the mechanism responsible for the increase in bile acid synthesis rates and the subsequent enlargement of bile acid pool sizes (BAPS) during the neonatal period, and how changes in circulating hormone levels might affect BAPS. In the hamster, pool size was low just after birth and increased modestly until 10.5 days postpartum (dpp). BAPS increased more significantly ( approximately 3-fold) between 10.5 and 15.5 dpp. An increase in mRNA and protein levels of cholesterol 7alpha-hydroxylase (Cyp7a1), the rate-limiting step in classical bile acid synthesis, immediately preceded an increase in BAPS. In contrast, levels of oxysterol 7alpha-hydroxylase (Cyp7b1), a key enzyme in bile acid synthesis by the alternative pathway, were relatively elevated by 1.5 dpp. farnesyl X receptor (FXR) and short heterodimeric partner (SHP) mRNA levels remained relatively constant at a time when Cyp7a1 levels increased. Finally, although simultaneous increases in circulating cortisol and Cyp7a1 levels occurred, precocious expression of Cyp7a1 could not be induced in neonatal hamsters with dexamethasone. Thus the significant increase in Cyp7a1 levels in neonatal hamsters is due to mechanisms independent of the FXR and SHP pathway and cortisol.

Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues.

The requirement for cholesterol is greater in developing tissues (fetus, placenta, and yolk sac) as compared to adult tissues. Here, we compared cholesterol-induced suppression of sterol synthesis rates in the adult liver to the fetal liver, fetal body, placenta, and yolk sac of the Golden Syrian hamster. Sterol synthesis rates were suppressed maximally in non-pregnant adult livers when cholesterol concentrations were increased. In contrast, sterol synthesis rates were suppressed only marginally in fetal livers, fetal bodies, placentas, and yolk sacs when cholesterol concentrations were increased. To begin to elucidate the mechanism responsible for the blunted response of sterol synthesis rates in fetal tissues to exogenous cholesterol, the ratio of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) to Insig-1 was measured in these same tissues since the ratio of SCAP to the Insigs can impact SREBP processing. The fetal tissues had anywhere from a 2- to 6-fold greater ratio of SCAP to Insig-1 than did the adult liver, suggesting constitutive processing of the SREBPs. As expected, the level of mature, nuclear SREBP-2 was not different in the fetal tissues with different levels of cholesterol whereas it was different in adult livers. These findings indicate that the suppression of sterol synthesis to exogenous sterol is blunted in developing tissues and the lack of response appears to be mediated at least partly through relative levels of Insigs and SCAP.

The liver plays a key role in whole body sterol accretion of the neonatal Golden Syrian hamster.

Neonates have a significant requirement for cholesterol. From -1 to 25 days of age, the liver accrues 6.9 mg cholesterol and the extra-hepatic tissues accrue 107.7 mg cholesterol in the hamster. It is currently unknown if each of these body compartments synthesizes their own cholesterol or if they have alternative source(s) of sterol. Using (3)H(2)O, in vivo hepatic sterol synthesis rates (per g liver per animal) increased between -1 and 5 days of age, decreased by 10 days of age, and increased again by 15 days of age. HMG-CoA reductase (HMGR) expression levels paralleled in vivo synthesis rates. Extra-hepatic sterol synthesis rates followed the same pattern as sterol synthesis rates in the liver. When sterol synthesis rates were converted to the mass of sterol synthesized per day, the liver synthesized 38.9 and the extra-hepatic tissues synthesized 63.9 mg cholesterol in the 26-day neonatal period. Comparing the amount of cholesterol accrued to that synthesized, one can conclude that the liver is a major source of sterol for the whole body during the neonatal period of the hamster. These results may help elucidate the cause(s) of reduced growth rates in neonates with liver disease or in neonates with compromised sterol synthesis rates.

Enhanced placental cholesterol efflux by fetal HDL in Smith-Lemli-Opitz syndrome.

Previous studies from this laboratory have shown that maternal-derived cholesterol can be effluxed from trophoblasts to fetal HDL and plasma. We had the opportunity to study for the first time the ability of HDL and plasma from a fetus with the Smith-Lemli-Opitz syndrome (SLOS) to efflux cholesterol from trophoblasts. It was unclear whether cholesterol could be effluxed to fetuses with SLOS since lipoprotein levels are often very low. To answer this question, cord blood was collected from the placentas of an SLOS fetus and unaffected fetuses just after delivery. Plasma cholesterol concentrations were very low in the affected fetus; cholesterol, 7-dehydrocholesterol, and 8-dehydocholesterol concentrations were 14.1, 4.5, and 5.2 mg/dl, respectively. The HDL from the fetal SLOS effluxed approximately 50% more cholesterol from a trophoblast cell line, were smaller in size, and had a lower cholesterol to phospholipid ratio as compared to HDL from unaffected fetuses or adults. Plasma from the SLOS fetus effluxed cholesterol to a similar percentage as unaffected fetal plasma or adult plasma, possibly due to fewer HDL particles as demonstrated in previous SLOS patients. These novel data demonstrate that the cholesterol-deficient SLOS fetus is able to obtain cholesterol from trophoblasts at a time when cholesterol is playing a critical role in development, and has implications for design of treatments for cholesterol deficiency syndromes as well as understanding of prenatal cholesterol transport in humans.

Effects of Multiple Freeze/Thaw Cycles on Measurements of Potential Novel Biomarkers Associated With Adverse Pregnancy Outcomes.

World-wide, millions of women enter preterm labor or have small newborns. Effective biomarkers are needed to identify women at risk for these adverse outcomes. A time and cost effective way to examine any potentially new biomarkers in samples collected during prior studies or trials that had been assayed for other metabolites would be highly useful. Thus, the current study aimed to determine if samples that had been previously thawed and re-frozen could be re-assayed for novel biomarkers, those being lipoprotein composition (sizing, proteome, lipids) and combined cholesterol and cytokine concentrations. Fasting blood was collected from 51 young non-pregnant women and plasma was analyzed for lipoprotein composition and cytokine concentrations after multiple freeze/thaw cycles in the cold or at room temperature and after being stored for 18 months. Plasma LDL-C, HDL-C, total cholesterol, and triglyceride concentrations decreased <6-7% (cholesterols) or <20% (triglyceride) after 7 thaws in the cold, 3 thaws at room temperature, and after 18 months of storage. As these decreases were less than day-to-day reported variation of lipids, they do not appear to be physiologically significant. Cytokine (IL-6, TNF α, IL-8, IL-1ß) and hsCRP concentrations decreased by 22%, 8%, 8%, 22%, and 35%, respectively; only IL-6, IL-1ß and hsCRP concentrations showed significant decreases greater than day-to-day variations of 20%. For measured triglyceride and cytokine, but not cholesterol concentrations, decreases with freeze/thaw cycles were greater when concentrations were elevated. Multiple thaws also led to changes in lipoprotein sizing, specifically to a shift from medium- and large-sized HDL particles to small-sized HDL particles and from large LDL to IDL. No changes occurred for VLDL particle numbers. Though particle sizes changed, the HDL proteome did not change with multiple thaw cycles or after long term storage. Overall, the results demonstrate that it is possible to use previously obtained frozen samples for plasma cholesterol and triglyceride levels and the lipoprotein proteome, and lipoprotein sizing and cytokine concentrations if one knows the history of the sample as changes should be relative to one another.