Ultraviolet Absorption Spectrophotometry Research Articles

Isolation and identification of 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2: new metabolites of 1,25-dihydroxyvitamin D2 produced in rat kidney.

Three new metabolites of vitamin D2 were produced in vitro by perfusing isolated rat kidneys with 1,25-dihydroxyvitamin D2. They were isolated and purified from the kidney perfusate by the techniques of methanol-methylene chloride lipid extraction and high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, and specific chemical reactions, the metabolites were identified as 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2. Both 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2 were also produced when a kidney was perfused with 1,24,25-trihydroxyvitamin D2. Thus, it becomes clear that 1,25-dihydroxyvitamin D2 is first hydroxylated at C-24 to form 1,24,25-trihydroxyvitamin D2, which is then further hydroxylated at C-28 and C-26 to form 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2, respectively. From several recent studies, it has been well established that 1,25-dihydroxyvitamin D3 is converted into various further metabolites in the kidney as a result of chemical reactions such as C-23, C-24, and C-26 hydroxylations, C-24 ketonization, and C-23:C-26 lactonization. From our study it is obvious that 1,25-dihydroxyvitamin D2 does not undergo all of the aforementioned chemical reactions except C-24 and C-26 hydroxylations. Also, our study indicates that C-28 hydroxylation plays a significant role in the further metabolism of 1,25-dihydroxyvitamin D2. Thus, for the first time, we describe a novel further metabolic pathway for 1,25-dihydroxyvitamin D2 in a mammalian kidney.

Quantitative characterization of procedures using ultraviolet and visible molecular absorption spectrophotometry

Abstract

Naturally occurring 24,25-dihydroxyvitamin D 3 is a mixture of both C-24 R and C-24 S epimers

Tritium-labeled 24,25-dihydroxyvitamin D 3 was prepared both in vitro, by using chick kidney homogenates, and in vivo in rats from [26,27- methyl- 3H]25-hydroxyvitamin D 3. These compounds were mixed with synthetic 24( R),25- and 24( S),25-dihydroxyvitamin D 3 converted to the corresponding trimethylsilyl ether derivatives, and analyzed by a high-pressure liquid chromatography procedure that separates the derivatized isomers. The tritium-labeled 24,25-dihydroxyvitamin D 3 derivatives were found to be a mixture of both the 24( R) and 24( S) epimers; the ratio was found to be 96.4:3.6 in chick kidney homogenates and 96.8:3.2 in the serum of rats under physiological conditions. In addition, nonradioactive 24,25-dihydroxyvitamin D 3 isolated from the serum of rats given large doses of vitamin D 3 was shown to be an 89.5:10.5 mixture of the 24( R) and 24( S) isomers. When 25-hydroxy-24-oxo-vitamin D 3 was utilized as a substrate, it was found to be more selectively reduced to 24( S),25-dihydroxyvitamin D 3 than 24( R),25-dihydroxyvitamin D 3 by the renal enzyme. The 24( S),25-dihydroxyvitamin D 3 has been identified by ultraviolet absorption spectrophotometry, cochromatography with an authentic standard, and mass spectrometry. The reduced metabolites of 25-hydroxy-24-oxo-vitamin D 3 were a 1:50 mixture of the 24( R) and 24( S) epimers. There are two known metabolic pathways leading to 24,25-dihydroxyvitamin D 3 from 25-hydroxyvitamin D 3; one is 24( R)-hydroxylation of 25-hydroxyvitamin D 3 and the other is reduction of 25-hydroxy-24-oxo-vitamin D 3. In contrast, 24( S),25-dihydroxyvitamin D 3 is produced only by reduction of 25-hydroxy-24-oxo-vitamin D 3 in the kidney. Therefore, naturally occurring 24,25-dihydroxyvitamin D 3 is a mixture of the 24( R) and 24( S) isomers, and not just the 24( R) isomer as reported previously.

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3.

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.

Synthesis of [10s(19)- 3H]-dihydrotachysterol 2 from ergocalciferol and preliminary investigations into its metabolic fate in rats

Dihydrotachysterol 2 (DHT 2), a 5,6- trans derivative of vitamin D 2, is very successfully used in the treatment of hypoparathyroidism and renal osteodystrophy. However, the metabolism and the action of DHT 2 are poorly understood. Investigations into metabolism of DHT 2 start at the synthesis of the radioactively labelled compound. This paper deals with a two-step synthesis of [10S(19)- 3H]-dihydrotachysterol 2 from vitamin D 2. Vitamin D 2 can be converted into 5,6- trans vitamin D 2 by iodination under irradiation and by triplet-sensitized isomerization. The first method led to unwanted side-reaction products which were difficult to separate from 5,6- trans vitamin D 2. Triplet-sensitized isomerization yielded merely 5,6- trans vitamin D 2 which could be separated from the residual starting material by chromatography on silica gel and recrystallization. [10S(19)- 3H]-Dihydrotachysterol 2 with a specific radioactivity of 56kCi/mol was prepared from 5,6- trans vitamin D 2 via partial, homogeneous catalytic reduction of the 10S(19) double bond with tritium gas. It was purified by high-performance liquid chromatography (h.p.l.c.) and characterized by chromatography and ultra-violet absorption spectrophotometry. The biological usefulness of the material was demonstrated in rats following intragastric administration. Blood was collected after 24 and 48 h and fractionation of serum lipids on h.p.l.c. showed 5 peaks of radioactivity.

Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios ( r < 0.1–0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.

Isolation and identification of 25-hydroxyvitamin D3-26,23-peroxylactone. A novel in vivo metabolite of vitamin D3.

A new vitamin D3 metabolite was isolated in pure form (18.2 micrograms) from the serum of rats given large doses (two doses of 26 mumol/rat) of vitamin D3. The new metabolite has been unequivocally identified as 3 beta, 25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-peroxylactone by ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, mass spectrometry, field desorption mass spectrometry, and specific chemical reaction with triphenyl phosphine. The stereochemical configuration at the C-23 and c-25 positions of the 25-hydroxyvitamin D3-26-23-peroxylactone was definitely determined to be the 23(S)25(R),25-hydroxyvitamin D3-26,23-peroxylactone is suggested for this metabolite. The isolation involved chloroform-methanol extraction and four column chromatographic procedures. The metabolite purification and elution position on these columns were followed by UV measurement at 264 nm. This metabolite was ultimately resolved from the previously known 25-hydroxyvitamin D3-26,23-lactone by high pressure liquid chromatography using a Zorbax Sil column. The 25-hydroxyvitamin D3-26,23-peroxylactone was converted upon storage at room temperature or -20 degrees C into the 25-hydroxyvitamin D3-26,23-lactone. Since under the conditions of this isolation only the 26,23-peroxylactone and no 26,23-lactone of 25-hydroxyvitamin D3 was present in the rat serum, this suggests that the 25-hydroxyvitamin D3-26,23-peroxylactone is the naturally occurring metabolite.

Isolation and identification of 23,25-dihydroxy-24-oxo-vitamin D 3: A metabolite of vitamin D 3

A new metabolite of vitamin D 3 has been isolated in pure form from incubations of rat kidney homogenates with 25-hydroxyvitamin D 3 [25-OH-D 3]. It was identified as 23,25-dihydroxy-24-oxo-vitamin D 3 [23,25(OH) 2-24-oxo-D 3] by means of ultraviolet absorption spectrophotometry and mass spectrometry. Also, 25-OH-D 3-26,23-lactone and 24R,25-dihydroxyvitamin D 3 were obtained from the same incubation mixtures. The enzyme activity responsible for the conversion of 25-OH-D 3 to 23,25(OH) 2-24-oxo-D 3 was induced by perfusion of the kidneys in vitro with 50 nM 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3].

Liquid chromatographic assay of phenothiazine, thioxanthene and butyrophenone neuroleptics and antihistamines in blood and plasma with conventional and radial compression columns and UV and electrochemical detection

An assay strategy for determining a wide range of phenothiazine, thioxanthene and butyrophenone neuroleptics and antihistamines both alone and in combination in blood and plasma is described. The general method employs liquid chromatography with both conventional and radial compression nitrile bonded columns. Detection is by ultraviolet absorption spectrophotometry or by amperometry depending on the concentrations to be measured. Ultraviolet absorption is suitable down to 10 ng/ml. Below this level amperometry is preferable. The various compounds are used as internal standards for each other. The lower limit of detection is approximately 0.1 ng ml −1 with a 10-ml sample. The within-run coefficient of variation is a maximum of 7.3%.

Isolation, identification, and biological activity of 23,25,26-trihydroxyvitamin D 3, an in vitro and in vivo metabolite of vitamin D 3

Kidney homogenates from vitamin D 3-supplemented chicks incubated with 25-hydroxyvitamin D 3 [25(OH)D 3] produce significant quantities of a new, unknown vitamin D metabolite. This metabolite was isolated in pure form from such incubation mixtures by using Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. This metabolite has been identified as 23,25,26-trihydroxyvitamin D 3 [23,25,26(OH) 3D 3] by loss of radioactivity from 25-hydroxy[23,24- 3H]vitamin D 3 and 25-hydroxy-[26,27-methyl- 3H]vitamin D 3, ultraviolet absorption spectrophotometry, mass spectrometry, and periodate cleavage oxidation followed by mass spectrometry. This same metabolite was also isolated from the serum of rats given large doses of vitamin D 3, and structurally characterized as 23,25,26-trihydroxyvitamin D 3. As yet, the stereochemistry at the C-23 and C-25 positions of the natural product remains unknown. A comparison of responses to a single dose level (500 ng) of 23,25,26(OH) 3D 3 or 25(OH)D 3 over 96 h in vitamin D-deficient rats indicated that the new metabolite had no capability to mediate bone calcium mobilization and that it was only weakly active in stimulating intestinal calcium transport.

Biliary metabolites of all-trans-retinoic acid in the rat: isolation and identification of a novel polar metabolite

The biliary metabolites from normal rats dosed with either pharmacological or physiological doses of all-trans-[11,12-3H2]retinoic acid were investigated. Biliary metabolites excreted during the first 24 h account for approximately 60-65% of the radiolabeled dose. A major polar metabolite was purified to homogeneity by using Sephadex LH-20 chromatography and several high-performance liquid chromatographic procedures. This metabolite was negatively charged as revealed by high-performance liquid chromatography on ion-exchange columns and accounts for 10% of the total biliary radioactivity (6% of the dose). The polar compound was positively identified by using Fourier transform proton nuclear magnetic resonance spectroscopy, high- and low-resolution mass spectrometry, fast atom bombardment mass spectrometry, ultraviolet absorption spectrophotometry, Fourier transform infrared spectroscopy, amino acid analysis, and chemical derivatization as 2-[8-[6-(hydroxymethyl)-2,6-dimethyl-3-oxo-1-cyclohexen-1-yl]-2,6- dimethyl-5,7-octadienamido]ethanesulfonic acid. The metabolic transformations required for the generation of this metabolite from all-trans-retinoic acid are the following: (1) allylic oxidation at carbon 4 of the cyclohexene ring to produce a 4-keto group, (2) hydroxylation of one of the methyl groups at carbon 1 of the cyclohexene ring, (3) saturation of the two terminal double bonds in the side chain, (4) loss of the terminal carboxyl group of the side chain via decarboxylation, and (5) conjugation of the resulting retinoid with taurine. To our knowledge, this metabolite represents the first taurine conjugate of a fat-soluble vitamin to be identified.

Hydrogen exchange kinetics of nucleic acids: Double and triple helices with Hoogsteen-type basepairs

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid)·poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine·uracil basepair has been found to exchange at a measurably slow rate, 0.023 s −1 (at 0°C), which is, however, much greater than that for a double-helix with the Watson-Crick type A·U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU)·poly(rA)·poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U·A·U base triplet have been found to exchange at a relatively slow rate, 0.0116 s −1 (at 0°C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U·A·U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3.

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.

1,25,26-trihydroxyvitamin D 3: Isolation, identification, and biological activity

A polar metabolite of vitamin D 3 has been produced in vitro from either 1,25-dihydroxyvitamin D 3 incubated with kidney homogenate from vitamin D-supplemented chickens or from 25,26-dihydroxyvitamin D 3 incubated with vitamin D-deficient chicken kidney homogenate. This compound was isolated in pure form and identified as 1,25,26-trihydroxyvitamin D 3 by ultraviolet absorption spectrophotometry and mass spectrometry. Furthermore, its periodate cleavage product comigrates with synthetic 1α-hydroxy-25-keto-27-norvitamin D 3 on high-performance liquid chromatography. The 1,25,26-trihydroxyvitamin D 3 is 0.1-0.01 as active as 1,25-dihydroxyvitamin D 3 in the stimulation of intestinal calcium transport and bone calcium mobilization.

Isolation and identification of 23,25-dihydroxyvitamin D3, an in vivo metabolite of vitamin D3.

Vitamin D supplemented rats produce a metabolite of 25-hydroxy[3 alpha-3H]vitamin D3 that is easily separated from known metabolites by using high-performance liquid chromatography. The production of this metabolite in vivo as well as 1,25-dihydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 26,23-lactone is largely if not totally eliminated by nephrectomy. Kidney homogenates from vitamin D supplemented chickens incubated with 25-hydroxyvitamin D3 produce significant quantities of the new, unknown metabolite. This metabolite was isolated in pure form from such incubation mixtures by using both straight-phase and reversed-phase high-performance liquid chromatography. This metabolite has been positively identified as 23,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry, mass spectrometry, and derivatization. This structure was confirmed by chemical synthesis of both C-23 stereoisomers. Although the natural product exactly comigrates with one of the synthetic isomers, the exact stereochemistry of the natural product remains unknown. It is possible that this new metabolite is an intermediate in the biosynthesis of 25-hydroxyvitamin D3 26,23-lactone.

The determination of lidocaine and benzocaine in isopropyl myristate

An in vitro membraneless drug release model utilizing an isopropyl myristate (ISM) sink as the acceptor phase has been used for the study of the release of lidocaine and benzocaine from aqueous Pluronic F-127 gels. The use of this system necessitated the development of a quantitative determination of lidocaine and benzocaine m isopropyl myristate solution. Extraction processes were developed to prepare the samples for the appropriate assay procedures: benzocaine by ultraviolet absorption spectrophotometry and lidocaine by gas chromatography. Direct assay of aqueous acid extracts of ISM sink solutions was not possible for benzocaine due to interfering UV absorbing substances. In the case of lidocaine, variable results were obtained from direct injections of the viscous ISM solutions into the GC column. In the latter case, however, direct assay was useful for the estimation of lidocaine concentrations, providing qualitative results for establishing appropriate concentrations necessary for a more complicated, but quantitative method based on a double extraction procedure.

Adsorption of nicotinic and isonicotinic acid derivatives by hydroxyapatite from aqueous solutions.

Adsorption of nicotinic and isonicotinic acid derivatives from aqueous solution by hydroxyapatite (HAP), the main component of tooth enamel, was investigated, and the effects of various factors on the adsorption were studied. These nicotinic and isonicotinic acid derivatives are similar in chemical structure to nicotine which is contained in tobacco, and which might be adsorbed by teeth during smoking. The amounts adsorbed at pH 6.0 and 7.0 at 37°C were determined by measurement of concentration differences (before and after adsorption). Six compounds, that is, nicotinic acid, isonicotinic acid, nicotinamide, isonicotinamide, nicotinic acid hydrazide, and isonicotinic acid hydrazide were used as drugs. All the drugs used were determined by ultraviolet absorption spectrophotometry. Adsorption isotherms were essentially of almost Langmuir type for every drug. The order of the amount adsorbed was 10-6 mol/g, and this value was smaller than that of chlorhexidine adsorption by HAP reported previously. It is suggested that the size of adsorbate molecules affects the adsorbability. The amounts adsorbed of isonicotinic acid derivatives were larger than those of the corresponding nicotinic acid derivatives. The order of the amounts adsorbed for both nicotinic and isonicotinic acid derivatives was as follows : acid>amide>hydrazide. This order was opposite to that of the solubility. The adsorption behavior of HAP was different from that of carbon black, a hydrophobic adsorbent. The amount adsorbed decreased upon addition of sodium chloride and sodium fluoride.

A versatile atomic-absorption spectrometric micro-attachment for cold-vapour atomic and gas-phase ultraviolet absorptiometric measurements

The applicability of a recently developed micro-attachment adaptable to common atomic-absorption spectrometers has been extended to include both cold-vapour atomic-absorption and gas-phase ultraviolet-absorption measurements. The cold-vapour atomic-absorption technique for the determination of total mercury in human whole blood and hair samples was applied by homogenizing the samples in a strongly alkaline medium and using stannous chloride-cadmium chloride mixture as reducing agent. Application of the system to the determination of volatile molecular species in aqueous solution by gas-phase ultraviolet-absorption spectrophotometry have also been studied, with NH3 as a model. This technique provides a simple, sensitive, yet selective method for the determination of ammoniacal and total nitrogen in aqueous samples and gives results comparable to those obtained by the standard distillation-titration method.

Isolation of a new metabolite of vitamin D produced in vivo, 1α,25-dihydroxyvitamin D 3-26,23-lactone

A new vitamin D metabolite was isolated in pure form from the blood of rats given oral doses of 50 μg/kg of 1α-hydroxyvitamin D 3. The isolation involved methanol-chloroform extraction and four successive column chromatographic procedures. A tentative structure of the metabolite is proposed on the basis of its column chromatographic behavior via mass spectrometry, ultraviolet absorption spectrophotometry, and as 1α,3β,25-trihydroxy-9,10 (19)-cholestatrieno-26,23-lactone. The trivial name 1α,25-dihydroxyvitamin D 3-26,23-lactone is suggested for this compound.

Hydrogen-deuterium exchange studies of proteins and nucleic acids

A kinetic study of the hydrogen exchange reaction of a protein or a nucleic acid can provide useful information on the structure and fluctuation of such a biological macromolecule. However, reactions faster than 10s are not easily traced by the methods used in earlier studies. Recent introduction of stopped-flow ultraviolet absorption and emission spectrophotometry into the hydrogen exchange studies of proteins and nucleic acids promises great development in this field. By this new technique we can now explore the millisecond region of the time-scale of hydrogen exchange kinetics. In this discussion we demonstrate some new aspects of the dynamic properties of proteins and nucleic acids on the basis of our results obtained by the stopped flow hydrogen exchange method.