A simple rapid separation of C1-esterase using an immunoadsorbent column.

An immunoadsorbent-antibody (egg albumin-Sepharose-antibody) column was found to be suitable for the rapid separation of C1-esterase from normal human serum. About 1.54 mg of C1-esterase, with a specific activity of 447 units/mg was obtained in voer 80% yield from the 20 ml of human serum.     

Spontaneous transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles

The transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles was studied by resonance energy transfer. The retinoic acid transfers spontaneously between single unilamellar vesicles with a first order rate constant of 9.6 s-1 at 15 degrees C and pH 7.4. At 30 degrees C, the transfer rate was 3.5 times faster than that at 10 degrees C. At pH 7.4, the transfer rate was almost 2 orders of magnitude faster than that observed at pH 1.6. Increasing the concentration of NaCl decreased the retinoic acid transfer rate significantly. Retinyl acetate transfers with a rate constant of 0.15 s-1, but no spontaneous transfer of retinyl palmitate was observed over 60 min. The evidence supports the proposal that retinoic acid and retinyl acetate transfer between single unilamellar vesicles occur via the aqueous phase. In contrast, no spontaneous transfer of retinyl palmitate was observed. However, linear free energy relationships and the thermodynamic parameters for retinyl acetate transfer permit the calculation of rate constant for retinyl palmitate transfer.     

The effects of nonadecafluoro-n-decanoic acid on serum retinol and hepatic retinyl palmitate hydrolase activity in male Sprague-Dawley rats

The effects of nonadecafluoro-n-decanoic acid (NDFDA) on serum retinol levels and hepatic retinyl palmitate hydrolase (RPH) activity were investigated in male Sprague-Dawley rats given a single intraperitoneal (IP) dose of 0, 50, or 100 mg/kg NDFDA and sacrificed at two, eight, or 11 days. Treated animals exhibited depressed serum retinol levels, lymphoid involution, and failure to gain weight in proportion to the dose. Hepatic RPH activities were depressed in both treatment groups at all time points and correlated with serum retinol levels. Hepatic retinol levels were also depressed by Day 11. Extraction of hepatic homogenates with acetone removed NDFDA and increased RPH activities twofold and threefold for the low- and high-dose groups, respectively. Analysis of partially purified RPH showed both NDFDA and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to be noncompetitive inhibitors: KI = 450 and 750 microM, respectively. We conclude that NDFDA causes a decrease in the mobilization of vitamin A from the liver by noncompetitive inhibition of RPH.     

Influence of dietary vitamins A and E on serum alpha- and gamma-tocopherols, retinol, retinyl palmitate and carotenoid concentrations in Humboldt penguins (Spheniscus humboldti)

Serum retinol, retinyl palmitate, beta-carotene, cryptoxanthin, lutein, alpha-tocopherol and gamma-tocopherol were measured in 18 captive Humboldt penguins (Spheniscus humboldti) prior to and following the removal of Columbia River (CR) smelt (Thaleichthys pacificus) from the diet. Dietary vitamin A was reduced from 59.8 to 13.5 IU g-1 (dry matter basis) when CR smelt was removed from the diet. Minimal changes were noted in dietary vitamin E. Serum samples Without-CR smelt had significantly lower circulating retinol (1.19 +/- 0.09 vs. 1.94 +/- 0.08 micrograms ml-1) and retinyl palmitate (0.033 +/- 0.012 vs. 0.105 +/- 0.004 microgram ml-1) compared to samples With-CR. The Without-CR smelt diet resulted in increased serum alpha-tocopherol from 26.4 +/- 0.94 to 39.1 +/- 3.72 micrograms ml-1. More serum samples taken Without-CR smelt had detectable levels of gamma-tocopherol than those With-CR smelt. Serum lutein was higher for the samples taken Without versus With-CR smelt. Serum cryptoxanthin did not differ. beta-Carotene was not detected. Data indicate that high levels of dietary vitamin A can affect circulating levels of retinol, retinyl palmitate and vitamin E. Thus, dietary vitamin A and the interrelationship between vitamins A and E should be considered when assessing captive penguins.     

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.     

A simple, rapid, and sensitive method of determining CSF IgG, using latex

A simple, rapid and sensitive method of determining cerebrospinal fluid IgG is presented. The procedure depends upon the fact that spinal fluid gamma globulin, and two of its components (IgG and IgM) will precipitate latex particles in proportion to their concentration, and that the optical density of the supernatant solution containing unprecipitated latex particles after centrifugation is inversely proportional to the concentration of these immunoglobulins. The method is sensitive to 50 nanograms, is inexpensive, requires 0.1 ml. or less of spinal fluid, and can be performed in minutes. Preliminary studies show that it compares favorably with results obtained by radial immunodiffusion.     

Developmental changes in vitamin A level and lack of retinyl palmitate in chick lungs

1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation.     

Vaginal hydrolysis of retinyl acetate: increase in plasma retinol and retinol binding protein in women with cervical dysplasias

We previously reported the clinical feasibility of a Phase I trial involving the topical administration of a RA gel applied cervicovaginally in women with mild or moderate cervical dysplasia. Now, we report hydrolysis and systemic absorption of the RA gel from the vagina. HPLC analysis of samples of residual gel obtained from the cervical canal after topical bolus application indicate that the RA undergoes prompt in vivo hydrolysis yielding retinol as a major metabolite. Venous blood samples of 41 subjects, who self-administered a RA gel, were analyzed for plasma retinol and RBP concentrations prior to and upon completion of a 7-day treatment course and upon return for follow-up examinations. An increase in both the concentrations of plasma retinol and RBP were detected after topical application of the RA gel. These elevated values receded after the gel administration was discontinued. No significant changes were observed in plasma retinol or RBP concentrations in placebo-treated subjects. The efficacy of RA as a chemopreventive agent in treating cervical dysplasias remains to be determined.     

A rapid and sensitive lipase assay using triethylaminoethyl cellulose columns

A procedure has been developed for the separation of labeled fatty acids from tri-, di-, and monoglycerides using small disposable columns of TEAE-cellulose. This procedure is used as the basis of a lipase assay which is rapid, sensitive and unaffected by wide variations in the composition of the reaction mixtures. 0.5 nmole [¹⁴C]oleic acid can be detected, and the entire procedure requires less than 3 min.     

A rapid and sensitive assay for chitinase using tritiated chitin

Radioactive chitin, prepared by acetylation of chitosan with tritiated acetic anhydride, was used as substrate in a rapid and extremely sensitive assay for chitinase. The procedure is based on the insolubility of chitin and the solubility in water of the reaction product, diacetylchitobiose. The course of the chitinase reaction is nonlinear, a result that cannot be attributed to an artifact of the method, to inhibition by product, or to instability of the enzyme. Some evidence points to structural heterogeneity of the substrate as a cause for this behavior. Reacetylated chitosan was also used as an adsorbent in the purification of chitinase with better results than with the previously used colloidal chitin.     

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL), and retinal (RAL) applicable to diverse biological samples with lower limits of detection of 0.7, 0.2, and 0.2 pmol, respectively, and linear ranges greater than 3 orders of magnitude. These assays function well with small, complex biological samples (10-20 mg tissue). Coefficients of variation range from 5.9 to 10.0% (intraday) and from 5.9 to 11.0% (interday). Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum). We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5 and 4.5 pmol and has a linear range and coefficient of variation similar to those of the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer.     

A rapid and sensitive assay of ADP glucose pyrophosphorylase using luciferase

A new, rapid and sensitive assay of ADPglucose pyrophosphorylase using the luciferaseluciferin system to quantify the reaction product, ATP, is described. The total assay time per sample is about 10 min. The sensitivity ranges from picomoles to nanomoles, and the crude enzyme extract from indicidual seeds of cereals can be assayed.     

A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format

Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.     

Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells

A study was conducted to determine the levels and distributions of retinoids, retinol-binding protein (RBP), retinyl palmitate hydrolase (RPH), cellular retinol-binding protein (CRBP), and cellular retinoic acid-binding protein (CRABP) in different types of isolated liver cells. Highly purified fractions of parenchymal, fat-storing (stellate), endothelial, and Kupffer cells were isolated in high yield from rat livers. The retinoid content of each fraction was measured by HPLC analysis. RBP, CRBP, and CRABP were measured by sensitive and specific radioimmunoassays, and RPH activity was measured by a sensitive microassay. The concentrations of each parameter expressed per 10(6) parenchymal or fat-storing cells were, respectively: retinoids, 1.5 and 83.9 micrograms of retinol equivalents; RBP, 138 and 7.4 ng; RPH, 826 and 1152 pmol FFA formed hr-1; CRBP, 470 and 236 ng; and CRABP, 5.6 and 8.7 ng. When these data were expressed on the basis of per unit mass of cellular protein, the concentrations of RPH, CRBP, and CRABP in the fat-storing cells, which contain 10-fold less protein than the large parenchymal cells, were seen to be greatly enriched over parenchymal cells. The parenchymal cells contained approximately 9% of the total retinoids, 98% of the total RBP, 90% of the total RPH activity, 91% of the total CRBP, and 71% of the total CRABP found in the liver. The fat-storing cells accounted for approximately 88% of the total retinoids, 0.7% of the total RBP, 10% of the RPH activity, 8% of the total CRBP, and 21% of the CRABP in the liver. The endothelial and Kupffer cell fractions contained very low levels of all of these parameters. Thus, the large and abundant parenchymal cells account for greater than 70% of the liver's RBP, RPH, CRBP, and CRABP; but the much smaller and less abundant fat-storing cells contain the majority of hepatic retinoids and greatly enriched concentrations of RPH, CRBP, and CRABP.     

A sensitive and rapid bioassay of homogalacturonan synthase using 2-aminobenzamide-labeled oligogalacturonides

Polygalacturonate 4-alpha-galacturonosyltransferase (GalA T) activity was detected in the microsomal fraction isolated from pumpkin (Cucurbia moschata Duchesne, cv. Tokyou-Kabocha) seedlings using UDP-GalA and 2-aminobenzamide (2AB)-labeled oligogalacturonides. A 2AB-labeled undecagalacturonide was elongated by the attachment of galacturonic acid (GalA) residues to give 2AB-labeled oligogalacturonides with a degree of polymerization (DP) between 12 and 17. Exogenous 2AB-labeled oligogalacturonide acceptors with a DP >3 are effective acceptor molecules for pumpkin GalA T.     

A fast, nondestructive purification scheme for prostaglandin H2 using a nonaqueous, bonded-phase high-performance liquid chromatography system

Arachidonic acid metabolism produces several biologically important compounds including the leukotrienes and prostaglandins. Prostaglandin H2 (PGH2) is the first metabolite in the arachidonic acid cascade leading to all other prostaglandins. Pivotal to our understanding of PGH2's biology is the ability to separate it in pure form from the numerous other arachidonic acid metabolites produced in a biological milieu. The extensive literature on PGH2 biology and metabolism has relied almost exclusively on the traditional method of separation using gravity flow silicic acid columns. In our hands, such PGH2 preparations were found to contain varying amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), PGE2, PGF2 alpha and other minor impurities as determined by further chromatographic and mass spectral analyses. Analytical separation of PGH2 and other arachidonic acid metabolites has been accomplished using reversed-phase HPLC. However, the labile nature of this molecule in aqueous systems makes such techniques unacceptable for preparative isolation of high purity PGH2 and has necessitated the development of a totally nonaqueous separation. To this end, we attempted several stationary phases and found that the cyano-bonded phase showed the best selectivity for resolving PGH2 from its major contaminants. Separations were performed on self-packed columns using a hexane-isopropanol gradient. Peaks were detected both by liquid scintillation counting and uv spectrophotometry (214 nm). Structure assignments were made by chromatographic comparison with authentic standards (PGF2 alpha, PGE2), biological activity (PGH2--platelet aggregation), and by ammonia direct chemical ionization mass spectrometry (HHT, hydroxy-5,8,10,14-eicosatetraenoic acid, PGH2, PGE2, PGF2 alpha). The latter technique, which by its very nature volatilizes all organic material in the sample, was particularly useful in determining not only that the PGH2 preparations were free from the aforementioned side products, but that they were also free from lipid, protein, and other potential residues frequently found in biological preparations.