High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Occurrence of vitellogenin in drone honeybees (Apis mellifica)

Summary

The occurrence of vitellogenin in adult haploid drones of the honeybee, Apis mellifica, was determined by sensitive immunotechniques, i.e. two-dimensional Immunoelectrophoresis and SDS-PAGE immunoblotting using a monospecific anti-vitellogenin-serum. In drones vitellogenin is one of the minor fractions of the hemolymph proteins. Genetic and regulatory aspects of vitellogenin synthesis in male bees are discussed.     

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes

A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females.In the three crustacean species studied (Uca pugilator, Cambarus clarkii, and Libinia emarginata), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk.It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.     

Development of male-incubated ovaries in the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PAGE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male-incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.     

Xenopus lipovitellin 1 is a Zn2+- and Cd2+-binding protein

This report discusses the identification of a Zn²⁺- and Cd²⁺-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with ⁶⁵Zn²⁺ or ¹⁰⁹Cd²⁺. The resulting autoradiograms showed binding of both radio-nuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co-purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (～115 kD) corresponded to reported values for lipovitellin 1 (111–121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis. © 1995 wiley-Liss, Inc.     

Vitellogenin-derived yolk proteins of white perch,Morone americana: purification,characterization, and vitellogenin-receptor binding

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.     

Biosynthesis,purification, and characterization of Aedes aegypti vitellin and vitellogenin

Vitellin, the major egg yolk protein, and vitellogenin, the hemolymph precursor of egg yolk protein, have been purified to apparent homogeneity from the mosquito Aedes aegypti. The purification procedure included chromatography on ion exchange, hydrophobic, and gel filtration columns. Vitellin and vitellogenin have a similar molecular weight (M_r 300,000) on gel filtration columns. However, the molecular weights of vitellin and vitellogenin, as determined from SDS electrophoresis, were 393,000 and 337,000, respectively. Vitellin in sodium dodecyl sulfate released six subunits of molecular weight 116,000, 83,000, 75,000, 54,000, 36,000, and 29,000, whereas vitellogenin released only three subunits (155,000, 120,000, and 62,000). The average molecular weights of vitellin and vitellogenin after gel filtration and SDS electrophoresis were 346,000 and 318,000, respectively. Vitellin has a high content of aspartic acid and glutamic acid, and a low content of histidine, methionine, cysteine, and tryptophan. Vitellin also contains 0.9% mol of glucosamine and no galactosamine. The isoelectric points of vitellin and vitellogenin are at pH 6.4 and 6.3, respectively. Aedes aegypti fat bodies incubated for short intervals in tissue culture medium in the presence of [³H]valine showed incorporation by radio-immunoprecipitation and SDS electrophoresis into three primary vitellogenin polypeptides of molecular weights (± SEM) 156,000 ± 4,000, 114,000 ± 5,000, and 62,000 ± 400 inside the fat body and 162,000 ± 3,000, 118,200 ± 2,000, and 63,000 ± 300 in the medium. These results suggest that the molecular weight of vitellogenin synthesized inside the fat body (M_r 332,000) remains unchanged when secreted into the hemolymph (M_r 343,000). The three vitellogenin subunits are processed by the ovary into six subunits which are then deposited in the yolk granules as vitellin.     

Isolation and identification of yolk proteins in Indian major carp,Labeo rohita

Two yolk proteins (YP1 and YP2) from the ovaries of Indian major carp, Labeo rohita were isolated by gel filtration and partially characterized by the use of hydroxyapatite ultrogel column in conjunction with native PAGE. On native PAGE YP1 gave a single protein band, whereas YP2 of gel filtration revealed the contamination of YP1, which was removed by adsorption chromatography on hydroxyapatite ultrogel and then the YP2 was the purified one as judged by electrophoresis. Both YP1 and YP2 also stained for lipid and contained alkalilabile phosphorus. Therefore, both yolk proteins were lipophosphoprotein. The molecular weights of YP1 and YP2 were 620 kDa and 225 kDa respectively as determined by gel filtration on Sepharose 4B. When YP1 and YP2 were compared in relation to some physicochemical characteristics with yolk proteins of other oviparous vertebrates including fish, they were lipovitellin like. Antiserum to YP2 crossreacted with YP2 and vitellogenin suggesting that YP2 was the cleaved product of vitellogenin. Anti-YP2 antiserum was not crossreacted with native YP1, whereas reduced and/or denatured YP1 was crossreacted indicating the presence of antigenic determinants in the inner core region of YP1 polypeptide.     

Lipovitellin 2β is the 31 kD Ni2+-binding protein (pNiXb) in Xenopus oocytes and embryos

An Ni²⁺-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with ⁶³Ni²⁺, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2β, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2β agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metalblot competition assay, the abilities of metal ions to compete with ⁶³Ni²⁺ for binding to pNiXb were ranked: Zn²⁺ ≈ Cu²⁺ ≈ Co²⁺ > Cd²⁺ ≈ Mn²⁺ > Sn²⁺. This study shows that Xenopus lipovitellin 2β is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo. © 1994 Wiley-Liss, Inc.     

Characterization of vitellogenin from white sturgeon,Acipenser transmontanus

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age. Correspondence to: C. A. Bidwell     

Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein.

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].     

Induction of vitellogenin synthesis in an Atlantic salmon (Salmo salar) hepatocyte culture: a sensitive in vitro bioassay for the oestrogenic and anti-oestrogenic activity of chemicals

A variety of organic compounds have been documented to bind to the oestrogen receptor and induce oestrogenic effects in different vertebrates. The presence of these environmental oestrogens or oestrogen mimics in the aquatic environment has been suspected of disrupting the normal endocrinology of wild populations of fish. In this study, induction of vitellogenin synthesis in primary hepatocytes from Atlantic salmon (Salmo salar) was optimized and validated as an oestrogenic in vitro bioassay using a sensitive capture vitellogenin enzyme-linked immunosorbent assay. After proper optimization (cell media supplements, cell density, temperature and exposure time), this assay gave a sensitive and reproducible response to both endogenous steroids (relative potency: 17β-oestradiol?oestriol>oestrone>17α-oestradiol) and a range of common oestrogen mimics (relative potency: ethynyloestradiol and diethylstilboestrol?genistein and zearalenone?bisphenol A and 4-t-octylphenol>4-n-nonylphenol and 2′-chloro,4-chloro-diphenyltrichloroethane (o,p′-DDT). However, the androgen testosterone and the putative oestrogen mimics dieldrin and toxaphene were not shown to be oestrogenic using this hepatocyte bioassay. Oestrogen-induced vitellogenin synthesis was efficiently inhibited by the anti-oestrogen ZM 189.154, suggesting that this bioassay may be used for testing both the oestrogenic and the anti-oestrogenic properties of chemicals.     

Polypeptide composition and biosynthesis of the yolk proteins in the firebrat,Thermobia domestica (insecta,thysanura)

Ion-exchange chromatography of crude ovarian extracts of the primitive insect Thermobia domestica allowed the separation, in native conditions, of major and minor vitellins of molecular weights of 300,000 and 430,000, respectively. Their polypeptide subunits were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunotransfer using an antiserum prepared against major vitellin. This protein was resolved into large (M_r 166,000–212,000) and small (around M_r 50,000) polypeptides. Minor vitellin, on the other hand, exclusively contained small polypeptides that are immunologically different from those of the major vitellin. Vitellogenin polypeptides from the hemolymph of mature females exhibited electrophoretic mobilities and immunological properties similar to vitellin polypeptides. Pulse-chase experiments showed that the female fat body synthesizes radioactive and immunoprecipitable proteins, whose polypeptide pattern is close to that of the major vitellogenin. However, part of the primary vitellogenic polypeptides, at M_r 210,000 and 212,000, is rapidly processed to M_r 176,000 and 182,000 subunits. These two polypeptides, as well as the precursors, enter into the composition of the major hemolymph vitellogenin. Finally, processing of the still uncleaved 210,000–212,000 polypeptides takes place in the ovary, which performs the same step of vitellogenin maturation as the fat body.     

Vitellin and vitellogenin in the soldier bug, Podisus maculiventris: identification with monoclonal antibodies and reproductive response to diet

A 171,000 M(r )polypeptide of Podisus maculiventris (Say) (Heteroptera: Pentatomidae) that constituted 16% of the protein in eggs also constituted up to 25% of the protein in hemolymph of fed females. It was identified as the major or sole apoprotein of vitellogenin. Eggs contained major polypeptides of 171, 106, and 51 kDa. The hemolymph polypeptide was identified with a polypeptide (vitellin) in egg extracts by comparing molecular weights, specificity of occurrence in fed females, and immunological reactivities. Females, starved for 5 days after eclosion to assure complete previtellogenic development, produced vitellogenin within a day after feeding on larval Galleria mellonella, and within 4 days after feeding on an artificial diet. Appearance of vitellogenin preceded ovarian growth by 2-3 days. Two monoclonal antibodies raised against egg proteins of P. maculiventris were selected for their strong reaction against egg extract and female hemolymph and null reaction against male hemolymph. Only one 170-kDa band in egg and hemolymph reacted with the antibodies on denaturing Western blots. These monoclonal antibodies are being used to develop an enzyme-linked immunosorbent assay (ELISA) to quantitate reproductive response of females to diets of differing quality.     

Electrophoretic patterns of yolk proteins throughout ovarian development and their relationship to vitellogenin in the rainbow trout,Oncorhynchus mykiss

• 1.1. Electrophoretic patterns of yolk proteins were investigated throughout ovarian development and their relationship to vitellogenin determined in a pulse-chase experiment with ³H-vitellogenin.
• 2.2. Using a radioimmunoassay for vitellogenin, vitellogenin/yolk protein products of vitellogenin were detected in follicles throughout ovarian development and in ovulated eggs.
• 3.3. The majority of yolk proteins in follicles measuring less than 1.0 mm in diameter appeared to be derived from sources other than vitellogenin. In contrast, in the larger follicles all of the major yolk proteins detected were derived from vitellogenin.
• 4.4. Pulse-chase with ³H-vitellogenin revealed that all of the major yolk proteins in 3.0 mm follicles were derived from vitellogenin. The major peptides eluted with molecular masses of 110 and 30 kDa under non-reducing conditions (these are very likely to represent lipovitellin 1 and lipovitellin 2), and 88, 22, 16, and 12 kDa under reducing conditions.
• 5.5. There were no apparent differences in the major yolk proteins in ovulated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis of these proteins had occurred during maturation and/or ovulation.

     

Validation of an ELISA for urinary dopamine: applications in monitoring treatment of dopamine‐related disorders

Dopamine is a catecholamine that serves as a neurotransmitter in the central and peripheral nervous system. Non‐invasive, reliable, and high‐throughput techniques for its quantification are needed to assess dysfunctions of the dopaminergic system and monitor therapies. We developed and validated a competitive ELISA for direct determination of dopamine in urine samples. The method provides high specificity, good accuracy, and precision (average inter‐assay variation < 12%). The analysis is not affected by general urinary components and structurally related drugs and metabolites. The correlation between ELISA and LC‐MS/MS analyses was very good (r = 0.986, n = 28). The reference range was 64–261 μg/g Cr (n = 64). Week‐to‐week biological variations of second morning urinary dopamine under free‐living conditions were 23.9% for within‐ and 35.5% for between‐subject variation (n = 10). The assay is applied in monitoring Parkinson's disease patients under different treatments. Urinary dopamine levels significantly increase in a dose‐dependent manner for Parkinson's disease patients under l ‐DOPA treatment. The present ELISA provides a cost‐effective alternative to chromatographic methods to monitor patients receiving dopamine restoring treatment to ensure appropriate dosing and clinical efficacy. The method can be used in pathological research for the assessment of possible peripheral biological markers for disorders related to the dopaminergic system.     

Studies on binding and uptake of vitellogenin by follicles of the tobacco hornworm,Manduca sexta

An in vitro system for the uptake of ¹²⁵l-vitellogenin (VG) or vitellin into isolated follicles of the tobacco hornworm, Manduca sexta, is described. After incubation with ¹²⁵l-VG, follicles were disrupted and the internal yolk contents separated from the follicle membranes. The results showed that ¹²⁵l-VG was associated principally with the membranes (92%) after incubation at 4°C. However, at 27°C, ¹²⁵l-VG was mainly in the yolk (92%). Furthermore, trypsin treatment removed approximately 70% of VG bound to the follicles at 4°C. Labeled VG was shown to bind to sonicated follicle membranes with high specificity and affinity (K_D ? 1.3 × 10^?8 M). This binding was sensitive to pH and calcium concentration. The total binding sites were estimated at 4 × 10¹⁴ sites/g of membrane protein. Competition studies showed that binding of ¹²⁵l-VG to follicle membranes was blocked by excess unlabeled vitellin and deglycosylated vitellogenin but not by lipophorin (the major hemolymph lipoprotein), microvitellogenin, a female-specific protein (M_r ～ 31,000) found in both hemolymph and eggs, and the smaller vitellogenin subunit, apovitellogenin-II (M_r ～ 45,000). These results suggest that selective uptake of M. sexta VG from the hemolymph involves binding to specific receptors located on the follicle membranes.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

Characterization of juvenile hormone-binding proteins of hemolymph of Locusta migratoria

The binding of juvenile hormone (JH) by components from hemolymph of adult female Locusta migratoria was characterized to establish whether hemolymph JH-binding proteins could be distinguished from a protein of fat body (BP-1) that may be a JH receptor. Hemolymph was analyzed by the hydroxyapatite assay, gel separation chromatography, polyacrylamide gel electrophoresis, and density gradient centrifugation. Three fractions that bound JH were separated from whole hemolymph by DEAE cellulose column chromatography, and these differed from all three cytosol-binding components. The major hemolymph component (H-A) showed relatively stable binding of JH, a slight loss of binding capacity after delipidation, and a K_d for JH-I of 16 nM. The K_ds for JH-l and JH-lll with unfractionated hemolymph were 26 and 42 nM respectively. The order of effectiveness of competitors for binding of [³H]JH-l was JH-lll > JH-l ? methoprene > hydroprene ? acids of methoprene and hydroprene. The data indicated that unlabeled JH-lll was bound more effectively than its radioactive counterpart. The sedimentation values determined by sucrose density gradient ultracentrifugation were 13-14 S for hemolymph, and the sedimentation value was not altered by the inclusion of 0.4 M KCl throughout the gradient. The data indicated that H-A resembled the specific JH carriers and differed from the putative receptor of fat body cytosol by several criteria.