Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Serum α- and γ-tocopherols, retinol, retinyl palmitate, and carotenoid concentrations in captive and free-ranging bottlenose dolphins (Tursiops truncatus)

Concentrations of retinol, retinyl palmitate, β-carotene, α-carotene, cryptoxanthin, lutein, lycopene, α-tocopherol, and γ-tocopherol were measured in blood samples collected from 15 captive and 55 free-ranging bottlenose dolphins (Tursiops truncatus). From June 1991 to June 1994, blood samples were collected from captive animals residing at two locations; at Seven Seas (Brookfield Zoo, Brookfield, IL) and Hawk’s Cay (Marathon Key, FL). Blood samples were collected from free-ranging animals from June 1991 to June 1996. Retinol levels were not significantly different between captive dolphin groups. However, Seven Seas animals had higher (P<0.01) serum retinol concentrations compared to free-ranging animals (0.061 vs 0.041 μg/ml). Retinyl palmitate was not detected in the serum of captive or free-ranging dolphins. Alpha-tocopherol levels were significantly (P<0.05) higher for Seven Seas dolphins (16.4 μg/ml) than for Hawk’s Cay (13.0 μg/ml) and free-ranging dolphins (12.5 μg/ml). Gamma-tocopherol concentrations were similar among captive and free-ranging dolphins. Free-ranging dolphins showed levels of circulating carotenoids (lutein and β-carotene) while the captive animals did not. Additional carotenoids (lycopene, α-carotene and cryptoxanthin) were analyzed but not detected in any samples. Serum vitamin differences between captive and free-ranging dolphins may reflect the natural diet or indicate some potential biological or nutritional status significance.     

Enhanced yolk testosterone influences behavioral phenotype independent of sex in Japanese quail chicks Coturnix japonica

Studies have demonstrated an effect of yolk testosterone levels on the physiology and behavior of nestling birds. In order to investigate this phenomenon experimentally in a precocial bird, we enhanced yolk testosterone, but within the physiological range, by injecting 50 ng testosterone in ethanol into Japanese quail Coturnix japonica eggs prior to incubation. The chicks hatching from these as well as from control eggs that had received the carrier-only or were left unmanipulated were subject to a number of behavioral tests from hatching to the age of 3 weeks. In addition, fecal samples were taken during a 90-min isolation period to determine a physiological response to a stressor. Experimental chicks performed a detour task faster and approached novel objects sooner than did the controls. Chicks from treated eggs took a longer time to start distress vocalizing and also produced less distress calls during open-field trials, took on average a larger number of trials for them to show tonic immobility and also excreted lower levels of corticosterone metabolites (BM) than did the controls. In response to a stressor, excreted BM was initially higher in the control chicks, as compared to the experimental birds. Induced behavioral effects were independent of sex with no sex treatment interactions found. In sum, experimentally enhanced testosterone levels in the eggs shifted individual behavioral phenotype towards "bold" or "proactive", irrespective of sex. We conclude that testosterone in the yolk influences the coping style of hatchlings and may be a potential means of maternal influence on offspring phenotype.     

Tissue-specific distribution of carotenoids and vitamin E in tissues of newly hatched chicks from various avian species

The aim of this study was to evaluate carotenoid and vitamin E distribution in egg and tissues of newly hatched chicks from wild mallard (Anas platyrhynchos), game pheasant (Phasianus colchicus), free-range guinea fowl (Numida meleagris), hen (Gallus domesticus) and domestic duck (Anas platyrhynchos) and intensively housed hens. Carotenoid concentrations in the egg yolk of free-range guinea fowl, pheasant and wild mallard were similar (61.3-79.2 microg/g). Egg yolks from ducks and intensively housed hens were characterised by the lowest carotenoid concentration comprising 11.2-14.8 microg/g. However, carotenoid concentration in eggs from free-range ducks and hens was less than half of that in free-range guinea fowl or pheasant. Depending on carotenoid concentration in the livers of species studied could be placed in the following descending order: free living pheasant>free-range guinea fowl>free-range hen>intensively housed hen>wild mallard>housed duck>free-range duck. The carotenoid concentrations in other tissues of free-range guinea fowl and pheasant were substantially higher than in the other species studied. Egg yolk of housed hens was characterised by the highest alpha- and gamma-tocopherol concentrations. In accordance with the alpha-tocopherol concentration in the egg yolk, the birds can be placed in the following descending order: intensively housed hen>wild mallard>free-living pheasant>free-range duck>free-range hen=free-range guinea fowl>housed duck. The main finding of this work is species- and tissue-specific differences in carotenoid and vitamin E distribution in the various avian species studied.     

Stability of individual carotenoids, retinol and tocopherols in human plasma during exposure to light and after extraction

We have modified gradient HPLC procedures for simultaneous quantification of retinol, γ-tocopherol, α-tocopherol, lutein/zeaxanthin, β-cryptoxanthin, trans-lycopene, cis-lycopene, α-carotene and β-carotene in 200-μl aliquots of human plasma. The photosensitivity of these analytes in plasma exposed to fluorescent lighting for up to 72 h was investigated and most were stable under these conditions. The stability of these analytes held in darkness at −20°C, 4°C or room temperature for up to 48 h after extraction from plasma was also investigated. Variability in measurement of most analytes was greater at room temperature than at 4°C or −20°C. There were statistically significant variations in the measured concentrations of some analytes in samples kept cold. However, the magnitude of these variations was small and of little biological significance, particularly over the first 24 h.     

Retinoid metabolism (LRAT,REH) in the yolk-sac membrane of Japanese quail eggs and effects of mono-ortho-PCBs

Retinoids stored in the avian egg are essential for normal development, however, laboratory and field experiments suggest that they are affected by environmental contaminants. Lecithin:retinol acyltransferase (LRAT) activity was detected in the microsomal fraction of the yolk-sac membrane of the Japanese quail at day 6 of development. LRAT activity was maximal at pH 7.0 having apparent kinetic parameters of K(m)=1.35 microM and V(max)=0.21 nmol/mg protein/h and was inhibited by the sulfhydryl modifying agent N-ethyl-maleimide. Retinol ester hydrolase (REH) activity in the microsomal fraction of the yolk-sac membrane was stimulated by the bile salt analogue 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane sulfonate and was maximal at pH 9.0 with apparent K(m)=77 microM and V(max)=34.3 nmol/mg protein/h. Injection of the PCB congener 2,3,3',4,4'-pentachlorobiphenyl increased both REH and LRAT activities, whereas 2,3,3',4-tetrachlorobiphenyl stimulated LRAT. Yolk retinol concentration and the molar ratio retinol:retinyl palmitate were lower in the exposed eggs. Yolk retinol concentration decreased as LRAT increased (R(2)=0.89) suggesting that certain PCB congeners may affect vitamin A mobilization in ovo by increasing LRAT activity in the yolk-sac membrane.     

Cleavage and blastoderm formation in normal and experimentally deformed naked eggs of a dipteran insect

Summary The eggs of the gall midgeHeteropeza pygmaea develop parthenogenetically inside of the mother larva. They lack a chorion and remain enveloped by the follicular epithelium. After experimental elimination of the follicular epithelium naked eggs are formed, which reach the blastoderm stage but remain spherical instead of assuming an elongated shape. To analyze this peculiar egg development and the roles of egg shape and envelope during development, the ultrastructure of cleaving normal and naked eggs was investigated. It was shown that the number of elements of Golgi apparatus and endoplasmic reticulum strongly increases during early cleavage. Their association with cleavage furrows and nuclei suggests that these organelles play a dominant role in membrane production. Egg yolk consists of lipids and glycogen, wheareas no proteins are found. Cleaving eggs contain numerous vesicles with lysosomal characteristics, indicating intense autophagic processes. Cleavage furrow formation occurs independently from the positioning of cleavage nuclei. The numerous microtubules, which are associated with cleavage furrows and nuclei and located in the egg periphery, the intercellular bridges, and in the central part of the egg, suggest that the cytoskeleton has an important role in cleavage furrow formation, blastoderm layer establishment, and yolk localization. Since these processes are accurately accomplished in naked spherical eggs, they can be considered as independent of normal egg shape and the follicular epithelium.     

卵黄抗体的分离提取和纯化方法

卵黄抗体性能稳定,具有较强抵抗热酸、碱的能力,室温下可保持6个月的活性,4℃下放置几年活性不减,易于大规模生产等许多优点,卵黄免疫球蛋白的纯化方法 和哺乳动物的纯化方法 不同,包括有机物沉淀法,有机溶剂抽提法,天然胶法和水稀释法等粗提方法;凝胶过滤层析,离子交换层析和亲和层析等精细纯化方法。本文就卵黄抗体的分离和纯化方法的优缺点作一比较。     

Plasma transport and tissue distribution of β-carotene, vitamin A and retinol-binding protein in domestic cats

The objective of this study was to determine retinol, retinyl esters and retinol-binding protein (RBP) as well as carotenoids in plasma, urine, liver and kidneys of randomly selected domestic cats. Retinol (240±64 ng/ml, mean±S.D.) represented one-third of total retinyl esters (736±460 ng/ml) in plasma. Retinyl esters were stearate, palmitate and oleate representing 61±6, 36±13 and 5±3% of total retinyl esters, respectively. In half of the cats, retinyl esters (22±21 ng/ml) were found in the urine. Vitamin A in the livers (4317±1956 μg/g) was significantly higher than in the kidney cortex and medulla (14.16±8.92 and 7.59±4.52 μg/g, respectively, both P<0.001). RBP was detected in the plasma but not in the urine. Immunoreactive RBP was observed in hepatocytes and in the cells of the proximal tubules. β-Carotene was present in plasma but never in tissues. The results show that similar to canines differences in vitamin A metabolism in cats are related to the occurrence of retinyl esters in plasma. They differ, however, with regard to the tissue distribution of β-carotene and the excretion of vitamin A in the urine.     

卵黄抗体在兽医寄生虫学中的应用

卵黄抗体是鸡产生的主要抗体,鸡被免疫后,IgY被持续地被合成、分泌到血液中,并被选择性地转移、富集到蛋黄中。母鸡产生的IgY可为它们的后代抵抗常见的禽类病原体提供有效的体液免疫保护。就有关寄生虫卵黄抗体的研制情况及其在兽医寄生虫病诊断、治疗等的应用情况做一综述。     

Antioxidant systems of the developing quail embryo are compromised by mycotoxin aurofusarin

The effects of aurofusarin in the quail diet on the antioxidant systems of the developing embryo are investigated. Thirty eight 45-day-old Japanese quails (Coturnix japonica) were divided into two groups and were fed on a corn-soya diet or the same diet supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin. Eggs obtained after 7 weeks of feeding were incubated. Samples of quail tissues were collected at day 17 of embryonic development and from day old hatchlings. Antioxidants and malondialdehyde were analysed by HPLC-based methods. Inclusion of aurofusarin in the maternal diet was associated with decreased concentrations of alpha- and gamma-tocopherols, alpha- and gamma-tocotrienols, retinol, lutein and zeaxanthin in egg yolk. The vitamin E (tocopherols and tocotrienols) concentration in the liver and yolk sac membrane (YSM) of the day 17 embryos and the hatchlings from aurofusarin-fed group was significantly decreased. Alpha-tocopherol concentration was also reduced in kidney, lung, heart, muscle and brain of day-old quails. In the liver of day-old quails, concentrations of lutein, zeaxanthin, retinol, retinyl linoleate, retinyl oleate, retinyl palmitate and retinyl stearate were also reduced. As a result of these diminished antioxidant concentrations, tissue susceptibility to lipid peroxidation was significantly increased. It is suggested that a compromised antioxidant system of the egg yolk and embryonic tissues could predispose quails to increased mortality at late stages of their embryonic development.     

Carotenoid discrimination by the avian embryo: a lesson from wild birds

The concentrations (μg/g wet yolk) of total carotenoids in eggs of the common moorhen (Gallinula chloropus), American coot (Fulica americana) and lesser black-backed gull (Larus fuscus), collected in the wild, were 47.5, 131.0 and 71.6, respectively. In contrast to data for eggs of the domestic chicken, β-carotene was a significant component in the yolks of these three wild species, forming 25–29% by wt. of the total carotenoids present. The concentration of total carotenoids in the livers of the newly-hatched chicks was 5–10 times higher than in the other tissues and β-carotene was again a major component, forming 37–58% of the hepatic carotenoids. In the newly-hatched gull, the proportions of both lutein and zeaxanthin were very low in the liver but high in the heart and muscle when compared with the yolk. By contrast canthaxanthin, echinenone and β-carotene were very minor constituents of heart and muscle when compared with their proportions in the yolk of the gull. The proportions of lutein and zeaxanthin in the liver of the newly-hatched coot and moorhen were also far lower than in the yolk whereas the liver was relatively enriched with β-cryptoxanthin, β-carotene and (in the moorhen) echinenone. The results indicate that avian embryos discriminate between different carotenoids during their distribution from the yolk to the various tissues.     

Cryopreservation of Spanish ibex (Capra pyrenaica) sperm obtained by electroejaculation outside the rutting season

The effectiveness of electroejaculation for obtaining Spanish ibex sperm samples for freeze preserving outside the rutting season was evaluated—the aim being to optimise biological resources for the establishment of germplasm banks. The effect of different egg yolk concentrations (6% or 12%, v/v) in diluents of different buffer composition (Tris-citric acid buffer or Tes-Tris buffer) on frozen-thawed samples of the above also investigated. Experiments were undertaken with six ibex males in February-May, and involved four different semen samples from each animal with four combination of extender, respectively: Tes-Tris-glucose (TTG)-6% egg yolk, TTG-12% egg yolk, Tris-citric acid-glucose (TCG)-12% egg yolk, TCG-6% egg yolk. The results show that electroejaculation is a useful way of obtaining sperm samples from Spanish ibex outside the rutting season (i.e., at a time coinciding with plasma testosterone levels of <0.4 ng/ml). According to the results of the eosin-nigrosin staining and the hypo-osmotic swelling test, the freezing-thawing process significantly reduced the viability and membrane integrity of the spermatozoa extended with TTG-6% egg yolk, TTG-12% egg yolk, and TCG-12% egg yolk, but did not affect these variables in spermatozoa extended with TCG-6% egg yolk. Therefore, the use of Tris-citric acid-based extenders containing low concentrations of egg yolk is recommended for cryopreserving Spanish ibex spermatozoa obtained by electroejaculation outside the rutting season.     

The influence of washing Spanish ibex (Capra pyrenaica) sperm on the effects of cryopreservation in dependency of the photoperiod

Extenders containing low concentrations of egg yolk are recommended for cryopreserving ibex spermatozoa. However, the phylogenetic relationship of the Spanish ibex (Capra pyrenaica) with domestic goats suggests that phospholipases in the seminal plasma may have a negative effect on the response to freezing-thawing when egg yolk-based diluents are employed. The aim of the current work was to determine how seminal plasma removal from Spanish ibex semen, collected by electroejaculation over a period of 1 yr, affects its response to freezing-thawing. Semen was collected from six adult ibexes maintained in captivity. The negative effects of freezing-thawing on the quality of sperm motility and on the integrity of the acrosome and plasma membrane were more serious in the nonwashed semen samples than in those from which the seminal plasma had been removed (P < 0.01, P < 0.05, and P < 0.05 respectively). The beneficial effect of removing the seminal plasma was particularly noticeable during the time of decreasing photoperiod. This suggests that ibex semen shows increased phospholipase activity during the rutting season.     

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species—the Spanish ibex—and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w v⁻¹), 2.2% citric acid (w v⁻¹), 0.6% glucose (w v⁻¹), 5% glycerol (v v⁻¹), and 6% egg yolk (v v⁻¹). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility (P < 0.001), membrane integrity (P < 0.001), and viability (P < 0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P = 0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.     

Egg yolk plasma can replace egg yolk in stallion freezing extenders

Hen egg yolk is normally used as a cryoprotective agent in semen freezing extenders, but its use has sanitary and practical disadvantages. Moreover the protection afforded by egg yolk has not yet been completely elucidated. The objective of this study was to compare the egg yolk plasma fraction to whole egg yolk in stallion freezing extender. Plasma contains mainly Low Density Lipoproteins (LDL), which are widely presumed to be the cryoprotective agent in egg yolk. Plasma can be produced on an industrial scale, sterilised by gamma-irradiation and incorporated in a ready-to-use extender (our ultimate objective). Plasma samples were subjected to different doses of gamma-irradiation (3, 5, 10 kGy) without dramatic chemical changes that may affect their cryoprotective properties. Stallion semen was frozen with whole egg yolk as a control and with sterilised egg yolk plasma. A fertility trial was conducted on a total of 70 mares' cycles. Fertility per cycle was 60% after insemination of semen frozen in our control extender containing egg yolk (EY), compared to 69% for the extender containing sterilised egg yolk plasma (EYP) (P > 0.05). Post-thaw motility and membrane integrity of spermatozoa were also analysed. Motility parameters were not significantly different between extenders except for the variable VAP (for EY versus EYP, VAP: 63 μm.s⁻¹ versus 59 μm.s⁻¹, a, b: P < 0.001; PROG: 41% versus 39%, RAP30: 56% versus 54%; RAP40: 51% versus 48%, P > 0.05). Membrane integrity was better preserved in EY than in EYP but the difference between extenders was small (P < 0.05). Our results demonstrated that sterilised egg yolk plasma has the potential to replace egg yolk in stallion freezing extender. This experiment led to the development of a ready-to-use extender called INRA-Freeze^® (IMV-Technologies, France).     

抗内毒素Fab’对严重烧伤早期肠源性内毒素血症小鼠NO、MDA与iNOS的影响及意义

目的：观察抗内毒素Fab’对严重烧伤早期肠源性内毒素血症小鼠肠组织中NO、iNOS、MDA水平的影响,探索防治烧伤脓毒症的新措施。方法：采用严重烧伤早期肠源性内毒素血症小鼠模型,分为烧伤组、治疗组及对照组,分别于6、12、24、48h4个时相点测定肠组织中NO、iNOS、MDA的浓度：结果：烧伤后肠组织中NO、iNOS、MDA水平均比正常对照组显著增高;治疗组肠组织中NO、iNOS、MDA水平较烧伤组显著降低。结论：抗内毒素Fab’能减轻内毒素对机体的损害,从而起到对严重烧伤早期肠源性脓毒症的防治作用。