Calf Liver Research Articles

Hypomethylation trends in the intergenic region of the imprinted IGF2 and H19 genes in cloned cattle

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/ H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/ H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/ H19 allelic expression. The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O’Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/ H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.

Differential induction of cytochrome P450 1A1 and 1B1 mRNA in primary cultured bovine hepatocytes treated with TCDD, PBDD/Fs and feed ingredients

This study investigates the dose-dependent expression of CYP1A1 and CYP1B1 in primary cultured bovine hepatocytes exposed to TCDD, several polybrominated dibenzo-p-dioxins and furans (PBDD/Fs) congeners and fish oil used as animal feed ingredients to identify their dioxin-like potentials. Hepatocytes were isolated from calf liver, cultured and treated for 24 h with the target compounds or extracts. Quantitative real time polymerase chain reaction analysis (qRT-PCR) showed that relative mRNA levels for CYP1A1 and CYP1B1 exhibited a dose-dependent induction by TCDD. The EC 50 of the TCDD concentration for CYP1A1 expression was approximately 4-fold less than that of CYP1B1. The estimated dioxin-like toxic potential of PBDD/Fs could be ranked in the following order: 2,3,7,8-TBDD > 1,2,3,7,8-PBDF > 2,3,4,7,8-PBDF > 1,2,3,6,7,8-HBDD. A good correlation was also observed in HRGC/HRMS-derived TEQs in fish oil samples and relative CYP1A1 mRNA induction in bovine hepatocytes treated with purified fish oil extracts. The data suggested that quantification of biomarker regulations in primary cultured hepatocytes could represent an effective tool for both the screening and study of various chemical entities in larger animals.

Organochlorine Pesticide and Polychlorinated Biphenyl in Calves from North-West Spain

The aim of the present study was to assess the levels of organic pollutants in cattle from the NW of Spain. The livers of 101 animals from two regions (Galicia and Asturias) were analysed for seven organochlorine pesticides (HCB, alpha-HCCH, u-HCCH, HEOD, DDT, TDE) and 34 PCBs congeners (8, 18, 28, 29, 31, 52, 77, 101, 105, 114, 118, 123, 126, 128, 138, 141, 149, 153, 156, 157, 167, 169, 170, 171, 180, 183, 187, 189, 194, 199, 201, 205, 206 and 209). The influence of different factors (type of farm and proximity to industrial areas) on pesticide accumulation in cattle was also studied. Overall, the frequency of occurrence and concentrations of OC pesticides and PCBs was low in calves from both regions. HEOD was detected most frequently and was found in more than half of the calves from each region. PCBs 141 and 153 were the most frequently detected PCB congeners but typically occurred in less than 20% of animals. The exposure of calves to the more abundant contaminants differed significantly between the regions and may be related to past usage patterns. Differences between farms in terms of their livestock management did not have a significant effect on pesticide accumulation by calves. The liver concentrations in calves were below maximum residues levels (RMLs) for human intake and there is no indication of any risk from these contaminants through eating calf liver.

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.

Changes in Hepatic Key Enzymes of Dairy Calves in Early Weaning Production Systems

The objective of the present study was to describe plasma hormonal and metabolite profile and mRNA expression levels and activities of the enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and acetyl-coenzyme A (CoA) carboxylase in the liver of male Holstein calves before (1 and 3wk of age) and after (8, 13, and 19wk of age) weaning at 6wk of age. The mean plasma concentration of acetate and β-hydroxybutyrate increased, and that of plasma lactate and nonesterified fatty acids decreased with week, particularly after weaning. Plasma glucose concentration was lowest at 8wk of age. The mean plasma concentration of insulin and glucagon did not change with time, and that of cortisol was greatest at 1wk of age. In the liver, enzyme activity of PC was greatest at 1wk of age and decreased with time. There was a significant relationship between the activity and the mRNA level for PC. Activity of PEPCK also decreased with week. Acetyl-CoA carboxylase activity tended to decrease with week, and activity at 13wk of age was lower than that at other times. Expression of PC mRNA, but not that of PEPCK and acetyl-CoA carboxylase α, decreased with week. We conclude that the hepatic gluconeogenic enzymes and acetyl-CoA carboxylase activities tend to decrease with age, reflecting changes in plasma metabolites in early weaning production systems.

소의 생리적 특성에따름 세포내 텔로미어 함량과 텔로머레이스 활성도 분석

Telomeres consist of TTAGGG tandem repeated DNA sequences with specific proteins and locate at chromosome ends. Telomeres are essential for chromosome stability and are related with cell senescence, apoptosis and cancer. Telomerase is a ribonucleoprotein which has a template for the synthesis of telomeric DNA. This study was carried out to analyze the amount of telomeric DNA and telomerase activity in cattle cells. Analysis of the quantity of telomere in lymphocytes was done at different ages, sex and among Korean cattle and Holstein breeds. The telomerase activity was also analyzed in liver, brain, heart, kidney, and testis tissues of fetal calf and of 18 month old cattle. The amount of telomeres in lymphocytes and other tissue cells was analyzed by Quantitative-Fluorescence in situ Hybridization (Q-FISH) technique using a telomeric DNA probe. Telomerase activity was analyzed by Telomeric Repeat Amplification Protocol assay (TRAP). The amount of telomeric DNA on the lymphocytes during the whole life span was decreased along with age. Quantity of telomeres in Korean cattle was significantly higher than that in Holstein breed. The amount of telomeric DNA in males was significantly higher than that in females. Telomerase activity was up-regulated in most bovine tissues during fetal stage, but was down-regulated in most tissues at mature 18 month age except the testis cells. This study indicates that the amount of telomeres and telomerase activity of cells can be used as an age marker or/and a physiological marker of cattle.

Cytochrome P450 inhibition profile in liver of veal calves administered a combination of 17β-estradiol, clenbuterol, and dexamethasone for growth-promoting purposes

The effects of the administration of a combination of 17β-estradiol (10 mg i.m. for three times at 17 days intervals), dexamethasone (4 mg/day for 6 days and 5 mg/day for further 6 days, dissolved in milk), and clenbuterol (20 μg/kg b.w./day, dissolved in milk, for the last 40 days before slaughtering) for growth-promoting (GP) purposes on liver drug metabolising capacity were studied in crossbred Friesian male calves. Compared to controls, liver preparations from GP-treated calves showed an overall reduction in the extent of the in vitro ability to metabolize testosterone and a number of substrates, most notably those associated with CYP 2C or CYP 3A, which also displayed a reduced expression on western blotting. By contrast, the tested hydrolytic and conjugative pathways were not significantly affected. As measured by northern blot, the lack of significant differences in CYP mRNA abundance point to a post-transcriptional effect of the GP combination. The remarkable involvement of the affected hepatic CYPs in the biotransformation of both steroid hormones and a large array of commonly used drugs may result in the further accumulation of undesirable residues in meat and offals of illegally treated calves.

Isolation and functional identification of a novel human hepatic growth factor: Hepatopoietin Cn

Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [(3)H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. HPPCn is a novel hepatic growth factor that plays a role in liver regeneration.

Intramuscular vaccination of young calves with aSalmonellaDublin metabolic-drift mutant provides superior protection to oral delivery

In homologous and heterologous challenge trials using calves <or= 6 weeks old, this comparative study investigated safety, in vivo behaviour and protective properties of oral and intramuscular vaccination with recently developed live attenuated Salmonella Dublin mutant N-RM25. Neither oral nor intramuscular vaccination produced unacceptable side effects. However, the vaccine strain was isolated for up to eight days from the faeces of orally vaccinated calves, but not intramuscularly vaccinated calves. Irrespective of the vaccination route, N-RM25 was isolated in low numbers (<or= 1 x 10(2) cfu/g) from the liver and spleen of calves euthanized at different time points post-vaccination. Vaccination by either route significantly reduced clinical signs and faecal shedding, prevented the development of systemic infection and protected calves from homologous lethal challenge conducted within 14 days post-immunisation. No challenge strain was isolated from major organs and the gut at 18 days post-challenge (except for a single mesenteric lymph node (MLN) specimen from the intramuscular group, but only following enrichment). Following heterologous challenge with a virulent Salmonella Typhimurium strain at 14 and 20 days post-immunisation, all vaccinated animals exhibited significantly fewer clinical signs and colonisation of the intestinal tract than non-vaccinated controls. When compared to oral vaccination, intramuscular vaccination significantly reduced the frequency of faecal shedding of S. Typhimurium (p = 0.0023) and markedly reduced colonisation ofMLN. The findings indicate that intramuscular administration of N-RM25 was safer in terms of environmental contamination by the vaccine and provided better early onset protection in young calves following both homologous and heterologous challenge.

Concentrations of Organotin Compounds in the Stranded Killer Whales from Rausu, Hokkaido, Japan

We measured the concentrations of butyltin (BT) and phenyltin (PT) compounds in blubber, liver, lung, and muscle of seven stranded killer whales (Orcinus orca) collected from Rausu, Hokkaido, Japan. BTs in blubber (n = 6), liver (n = 4), lung (n = 1), and muscle (n = 4) of adult whale were in the range of 37-90, 385-676, 15, and 26-53 microg kg(-1) wet weight, respectively. Concentrations of PTs in blubber, liver, lung, and muscle were <1 to 58, <1 to 14, 7 and <1 to 4 microg kg(-1) wet weight, respectively. Detected frequencies of PTs were low in all tissues. Higher percentages of monobutyltin (MBT) in blubber, lung, and muscle were observed, while the percentage of dibutyltin (DBT) was high in liver. Detected frequencies of triphenyltin (TPT) were highest among tissues. Total BT concentrations in blubber and liver of a whale calf were lower than those in adult whales. MBT and DBT in the liver of the calf were the same (42%). MBT in blubber was the dominant compound among BTs.

Low-dietary protein intake induces problems with glucose homeostasis and results in hepatic steatosis in heavy milk-fed calves

We studied effects of protein intake at two protein-free energy intake levels on plasma glucose and insulin concentrations, urinary glucose excretion and on liver and intestinal fat content in milk-fed veal calves. Two experiments were performed at body weights (BW) of 80–160 kg (mean 120 kg; Exp. 1) and 160–240 kg (mean 200 kg; Exp. 2). In each experiment, 36 calves were allocated to one of six protein intake levels, at each of two energy intake levels. Digestible protein intakes ranged between 0.90 and 2.72 g nitrogen (N)/(kg BW 0.75 × d) in Exp. 1 and between 0.54 and 2.22 g N/(kg BW 0.75 × d) in Exp. 2. The two energy intake levels were kept constant on a protein-free basis and were 663 and 851 kJ/(kg BW 0.75 × d) in Exp. 1 and 564 and 752 kJ/(kg BW 0.75 × d) in Exp. 2. Blood samples were taken between 5 and 6 h post-feeding at 14-d intervals until calves reached target BW, and liver fat mass was determined at slaughter. Urinary glucose excretion was quantified at 120 and 200 kg BW in Exps. 1 and 2, respectively. Increased protein-free energy intake increased plasma glucose concentrations and urinary glucose losses in 200 kg calves, but not in 120 kg calves. Increasing protein intake decreased plasma glucose, urinary glucose and plasma insulin in both experiments. Liver fat content decreased with increasing protein intake. In conclusion, long-term low-dietary protein intake increased hyperglycemia, hyperinsulinemia, glucosuria and hepatic steatosis in heavy milk-fed calves, likely associated with increased insulin resistance.

Spatial Inhomogeneity of Tissue Thermal Parameter of Ebbini's Model and Its Dependency on Temperature

The thermal parameter k of Ebbini's model (i.e., expressed by the temperature coefficients of ultrasound speed and volume change) for temperature measurement is experimentally investigated using a fresh calf liver specimen in vitro. It is found that k is spatially inhomogeneous and dependent on temperature and thermal exposure time. It is also clarified that it is difficult to accurately measure a large temperature change and a high temperature using Ebbini's model. To enable the planning of thermal treatment, we need to evaluate temperature distribution and reconstruct tissue thermal conductivity, capacity and diffusivity by updating k or spatial mean k using temperature references (thermocouples).

Quantification of cadmium and lead in offal by SF-ICP-MS: Method development and uncertainty estimate

Offal of bovine, ovine and porcine are able to accumulate potentially toxic heavy metals, such as Cd and Pb, posing a risk for human health. For this reason, the Commission Regulation no. 466/2001 provided the maximum admitted levels for these metals in this kind of matrix (Cd, 500 ng g −1 in all kinds of offal; Pb, 500 ng g −1 in liver and 1000 ng g −1 in kidney). A method based on sector field inductively coupled plasma mass spectrometry for Cd and Pb quantification in calf liver was developed and further applied to offal of different animals. The uncertainty of measurements was calculated according to the Eurachem/Citac Guide. The method LoDs and LoQs were 3.5 and 11 ng g −1 for Cd, and 2.0 and 6.0 ng g −1 for Pb. The repeatability and the intra-laboratory reproducibility showed relative standard deviations equal to 2.25% and 1.99% for Cd and 1.49% and 6.55% for Pb. Relative expanded uncertainties at the mean value in calf liver were 4.74% for Cd and 13.8% for Pb. In Italian offal the following concentration intervals were found (in ng g −1): (i) Cd: calf, from <3.5 in spleen and lung to 96.4 in kidney; lamb, <3.5 in all offal; pig, 114 in liver and (ii) Pb: calf, from 4.03 in lung to 31.8 in liver; lamb, from 4.71 in heart to 279 in liver; pig, 9.19 in liver.

Interaction of Immobilized Human Exportin-t with Calf Liver tRNA

The binding of tRNA species from calf liver to immobilized exportin-t in the presence of ran•GppNHp was examined by affinity chromatography. We observed different eluting behaviors of individual tRNAs. After separating tRNAs on a two-dimensional polyacrylamide gel, the positions of 7 selected tRNAs were identified by Northern hybridization and their relative affinities to immobilized exportin-t•ran•GppNHp complex were estimated in the order of tRNALeuCAG > tRNASerGCU, tRNALeuCAA, tRNASerUGA > tRNASerAGA, tRNALeuAAG > tRNAArgACG. We propose that exportin-t preferentially binds and exports those tRNAs that are rapidly consumed by the protein synthesis machinery.

Protein Synthesis in Jejunum and Liver of Neonatal Calves Fed Vitamin A and Lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n=6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[13C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (APFree) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APEPla) and in the CO2 of exhaled air (APEEx). The APE, APFree, and FSR in the jejunum and liver did not differ significantly among groups. The APEEx increased, whereas APEPla decreased over time, but there were no group differences. Correlations were calculated between FSRJej and histomorphometrical and histochemical data of the jejunum, and between FSRLiv and blood metabolites. There were negative correlations between FSRLiv and plasma albumin concentrations and between FSRJej and the ratio of villus height:crypt depth, and there was a positive correlation between FSRJej and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSRJej was correlated with histomorphometrical traits of the jejunum and FSRLiv was correlated with plasma albumin concentrations.

The estimation of elasticity and viscosity of soft tissues in vitro using the data of remote acoustic palpation

The presented study revealed the significant dependence of displacement magnitude and strain relaxation on phantom elasticity and viscosity. It has been shown that simultaneous analysis of temporal behavior and magnitude of shear strain induced by the radiation force of focused ultrasonic beam gives the necessary data for quantitative estimation of tissue shear modulus because of the known functional dependencies of displacement on local viscosity and elasticity. As a result, the simplest calibration procedure of acoustic radiation force-based methods is performed and algorithm for separate reconstruction of tissue elasticity and viscosity is proposed. These findings were tested, in particular, using the data obtained for specially prepared phantoms containing calf liver and muscle tissue in vitro. The observed complex character of shear strain relaxation and noise in some tissue phantoms and tissues in vitro reduces the preciseness of viscoelastic properties estimation. (E-mail: barannik@niiri.kharkov.com)

Expression of Nuclear Receptor and Target Genes in Liver and Intestine of Neonatal Calves Fed Colostrum and Vitamin A

Nuclear receptors (NR), including retinoic acid and retinoid X receptors (RAR, RXR), pregnane X receptor (PXR), constitutive androstane receptor, and peroxisome proliferator-activated receptor (PPARα) modify the expression of other genes, such as cytochrome p450 enzymes (CYP), sulfotransferases (SULT), and UDP glucuronosyl transferases (UGT). Nuclear receptor expression is influenced by exposure to ligands (e.g., vitamin A). We tested the hypothesis that vitamin A feeding influences the expression of hepatic and intestinal NR and their target genes and that colostrum or formula feeding influence these traits differently. Calves (n = 7/ group) were fed colostrum (CO) or a milk-based formula with or without vitamin A (FA, FO, respectively) for 4 d and were euthanized on d 5, followed immediately by tissue collection. Thereafter, RNA was extracted and gene expression quantified by real-time reverse transcription-polymerase chain reaction. Expression relative to housekeeping genes of mRNA was profiled for NR, CYP, SULT, and UGT enzymes. Hepatic mRNA levels of RARβ and CYP26 were higher in FA than FO cows; expression of CYP2E1, CYP2C8, CYP26, and UGT1A1 was higher in CO than FO cows; and expression of CYP2E1, UGT1A1, and p450 reductase was higher in CO than FA. In colon tissue, abundance of RXRα mRNA was lower in FO than CO, and CYP2B6 expression was lower in FO than in CO and FA. In jejunal tissue, there were no significant differences in gene expression among groups. In conclusion, effects of vitamin A feeding were limited, but colostrum feeding had several selective effects on expression of nuclear receptors and target genes.

The C-terminal Appended Domain of Human Cytosolic Leucyl-tRNA Synthetase Is Indispensable in Its Interaction with Arginyl-tRNA Synthetase in the Multi-tRNA Synthetase Complex

Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.

NEONATAL HEMOCHROMATOSIS (NH) DUE TO GESTATIONAL ALLOIMMUNITY: DETECTION OF MATERNAL ALLOANTIBODY AND PREVENTION OF RECURRENCE

Background: NH is a severe fetal liver disease. We hypothesized that NH is an alloimmune gestational disease and showed that treatment with high-dose IVIG during gestation prevents lethal recurrence (Lancet 2004;364:1690-8). The aims of this study were: 1. Identify the fetal target of alloimmunity in order to develop a serologic test for alloantibody; and 2. Extend the trial of gestational treatment in order to further determine its effectiveness. Methods and Results: Subjects were women whose last pregnancy ended in a bona fide case of NH; controls were unaffected women and spouses of subjects. IgG isolated by protein-A sepharose binding was tested for immunoreactivity with fetal liver proteins by western blot and 2-D PAGE immunoblot. IgG from NH subjects consistently reacted at high dilution with a specific protein in fetal (but not adult) human, mouse and calf liver, whereas control IgG at low titer at most weakly detects this protein, which is tentatively identified as a fetal isoform of hemeoxygenase-1 (HO-1). This protein isolated from fetal calf liver is being used to develop a sandwich immunoassay for quantitative, high throughput determination of alloantibody. Subjects were offered gestational treatment (IVIG 1g/kg weekly from 18 wks gestation) in subsequent pregnancies. To date, 29 subjects have enrolled. All 25 babies born to date survived with medical care alone (p < 0.00001 versus previous gestations); 5 women are currently receiving treatment; and 1 subject suffered miscarriage at 18 weeks before treatment began. We became aware after the fact that a woman beginning off-protocol treatment at 24-25 weeks had a severely affected baby who died. She subsequently tested strongly positive for alloantibody. Conclusions: NH is often the result of maternal alloimmunity against fetal liver and recurrence can be prevented by high-dose IVIG during gestation, beginning at 18 weeks or earlier. We speculate that depletion of fetal hepatic HO-1 activity results in loss of cellular antioxidant and sensitivity to iron-induced oxidant injury.

Vitamin A content (retinol and retinyl esters) in livers of different animals

In the present study, 90 animal livers of five different species (pig, cattle, calf, chicken, turkey) were examined for their vitamin A contents. The investigation of extracted vitamin A included all- trans retinol, retinyl palmitate, stearate, oleate and linoleate, expressed as retinol equivalents (RE). The separation of the various chemical forms was done using HPLC. The liver vitamin A contents ranged between 6.5 and 18.9 mg RE/100 g in pigs, from 1.1 to 6.7 mg RE/100 g in cattle and from 1.6 to 16.6 mg RE/100 g and 2.7 to 21.5 mg RE/100 g in chickens and turkeys, respectively. The livers of calves contained the smallest amount of vitamin A, with variation from 1.3 to 3.2 mg RE/100 g. Retinyl palmitate was the predominant form of vitamin A in the livers of investigated animals and contributed about 40% (avids) up to 75% (calf) of the total liver vitamin A contents. The results indicated that the lower levels of animal liver vitamin A, observed in our study, could be a result of small-structured agriculture in Austria. The variations of liver vitamin A concentrations among the species were a result of differences in race, age and the different feeding regimen.