Synergistic effect of low K and D vitamin status on arterial stiffness in a general population

Both vitamins K and D are nutrients with pleiotropic functions in human tissues. The metabolic role of these vitamins overlaps considerably in calcium homeostasis. We analyzed their potential synergetic effect on arterial stiffness. In a cross-sectional study, we analyzed aortic pulse wave velocity (aPWV) in 1023 subjects from the Czech post-MONICA study. Desphospho-uncarboxylated matrix γ-carboxyglutamate protein (dp-ucMGP), a biomarker of vitamin K status, was measured by sandwich ELISA and 25-hydroxyvitamin D₃ (25-OH-D₃) by a commercial immunochemical assay. In a subsample of 431 subjects without chronic disease or pharmacotherapy, we detected rs2228570 polymorphism for the vitamin D receptor. After adjustment for confounders, aPWV was independently associated with both factors: dp-ucMGP [β-coefficient(S.E.M.) = 13.91(4.87); P=.004] and 25-OH-D₃ [0.624(0.28); P=.027]. In a further analysis, we divided subjects according to dp-ucMGP and 25-OH-D₃ quartiles, resulting in 16 subgroups. The highest aPWV had subjects in the top quartile of dp-ucMGP plus bottom quartile of 25-OH-D₃ (i.e., in those with insufficient status of both vitamin K and vitamin D), while the lowest aPVW had subjects in the bottom quartile of dp-ucMGP plus top quartile of 25-OH-D₃ [9.8 (SD2.6) versus 6.6 (SD1.6) m/s; P<.0001]. When we compared these extreme groups of vitamin K and D status, the adjusted odds ratio for aPWV≥9.3 m/s was 6.83 (95% CI:1.95–20.9). The aPWV was also significantly higher among subjects bearing the GG genotype of rs2228570, but only in those with a concomitantly poor vitamin K status. In conclusion, we confirmed substantial interaction of insufficient K and D vitamin status in terms of increased aortic stiffness.     

Metabolism of 1alpha-hydroxyvitamin D3 to 1alpha, 25-dihydroxyvitamin D3 in perfused rat liver.

The metabolism of 1α-hydroxyvitamin D₃ (1α-OH-D₃) was studied in rat liver perfused with [³H]-1α-OH-D₃. [³H]-1α-OH-D₃ was converted very rapidly to a more polar metabolite, which was identified as 1α,25-dihydroxy-vitamin D₃ [1α,25-(OH)₂-D₃] by co-chromatography with synthetic 1α,25-(OH)₂-D₃ as well as by gas chromatography-mass spectrometry. [³H]-1α,25-(OH)₂-D₃ appeared in the perfusate as early as 20 min after addition of [³H]-1α-OH-D₃, and its level in the perfusate increased linearly for at least 120 min. These data strongly indicate that 1α-OH-D₃ is metabolized to 1α,25-(OH)₂-D₃, which exerts biological effects on bone and intestine.     

Bone resorbing activities of 24-hydroxy stereoisomers of 24-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

R and S isomers of 24-OH-D₃ and 24,25-(OH)₂D₃ were tested for their effects on bone resorption $\text{in vitro}$. 24(R), 25-(OH)₂D₃ was more active than 24(S),25-(OH)₂D₃. Likewise, 24(R)-OH-D₃ was more active than 24(S)-OH-D₃. The bone resorbing activity of 24(R)-OH-D₃ was equivalent to that of 25-OH-D₃; 24(R),25-(OH)₂D₃ was somewhat less potent. The results indicate that there is discrimination between the isomers of these compounds at the level of the responding tissue.     

Circadian rhythm of 1 alpha,25-dihydroxyvitamin D3 production in egg-laying hens.

The activity of renal 25-hydroxyvitamin D₃(25-OH-D₃)-1α- and 24-hydroxylase and the plasma concentrations of vitamin D metabolites were investigated in relation to the ovulatory cycle in egg-laying hens. The time after ovulation was estimated from the position of the egg in the oviduct and the dry weight of the egg-shell. The $\text{in}\text{vitro}$ renal 25-OH-D₃-1α-hydroxylase activity was significantly enhanced 14–16 hr after ovulation, whereas 25-OH-D₃-24-hydroxylase activity remained unchanged. The plasma level of 1α,25-dihydroxyvitamin D [1α,25-(OH)₂-D] was also increased 14–16 hr after ovulation in accord with the enhancement of the renal 1α-hydroxylase activity. The plasma level of 24,25-dihydroxyvitamin D did not change during the ovulatory cycle. These results strongly suggest that 1α,25-(OH)₂-D₃ production in the kidney varies in a circadian rhythm during the ovulatory cycle in egg-laying hens.     

High-pressure liquid chromatographic method for the determination of 25-hydroxycholecalciferol in cow plasma

A high-pressure liquid chromatographic (hplc) procedure was developed for the determination of 25-hydroxycholecalciferol (25-OH-D₃) in cow plasma or serum. The procedure involved extraction with an ethanol-ethyl ether mixture, separation of the aqueous phase, solvent partitions, column chromatography on silica gel, and, finally, determination by reversed phase hplc on a C₁₈-bonded microparticulate silica column. The identity of the drug in the extract was confirmed by comparison with a standard by liquid, thin-layer, and gas-liquid chromatography as the free steroid and the heptafluorobutyrate and by the uv spectra and also from the mass spectrum of the heptafluorobutyrate. Twenty-four samples from cows on normal diet (dry, lactating, and pregnant) were analyzed. The normal circulating levels of 25-OH-D₃ ranged from 40 to 58 ng/g; mean 48 ± 5.0 ng/g. The procedure was used to analyze a limited number of human and hog samples. Human serum contained 10–20 ng/g which was in agreement with literature values. Hog serum contained 18 ng/g.     

Cross-talk among structural domains of human DBP upon binding 25-hydroxyvitamin D

Serum vitamin D-binding protein (DBP) is structurally very similar to serum albumin (ALB); both have three distinct structural domains and high cysteine-content. Yet, functionally they are very different. DBP possesses high affinity for vitamin D metabolites and G-actin, but ALB does not. It has been suggested that there may be cross-talk among the domains so that binding of one ligand may influence the binding of others. In this study we have employed 2-p-toluidinyl-6-sulfonate (TNS), a reporter molecule that fluoresces upon binding to hydrophobic pockets of DBP. We observed that recombinant domain III possesses strong binding for TNS, which is not influenced by 25-hydroxyvitamin D₃ (25-OH-D₃), yet TNS fluorescence of the whole protein is quenched by 25-OH-D₃. These results provide a direct evidence of cross-talk among the structural domains of DBP.     

A specific binding protein for 1,25-dihydroxyvitamin D3 in rat intestinal cytosol.

In the presence of 0.3 M potassium chloride and 0.5 mM dithiothreitol, rat intestinal cytosol contains two binding proteins for 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)¹ having sedimentation coefficients of 3.2S and 5–6S. The 3.2S protein is specific for 1,25-(OH)₂D₃ as determined by competition analysis, whereas the 5–6S protein binds 25-hydroxyvitamin D₃ (25-OH-D₃) exclusively.     

The site of 1α,25-dihydroxyvitamin D3 production in pregnancy

Metabolism of 25-hydroxyvitamin D₃ (25-OH-D₃) in pregnancy was investigated $\text{in}\text{vitro}$ in New Zealand White rabbits fed a rabbit chow. Kidney homogenates from pregnant mothers and fetuses were separately incubated with [³H]-25-OH-D₃. The homogenates from fetuses produced significant amounts of $\text{[}{}^3\text{H]-1α,25-dihydroxyvitamin}$ D₃ [1α,25-(OH)₂-D₃] from its precursor, while those from mothers predominantly produced [³H]-24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃]. The identity of the radioactive metabolites produced from [³H]-25-OH-D₃ was established by periodate cleavage and comigration with synthetic 1α,25-(OH)₂-D₃ or 24,25-(OH)₂-D₃ on high pressure liquid chromatography. These results clearly indicate that the fetal kidney is at least one of the sites of 1α,25-(OH)₂-D₃ synthesis in pregnancy.     

Early effect of 25-hydroxycholecalciferol (25-OH-D3) and 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) on the ability of parathyroid hormone (PTH) to elevate cyclic AMP of intact bone cells.

25-OH-D₃ and 1,25-(OH)₂-D₃ had no effects by themselves on the cyclic AMP levels of isolated bone cells but enhanced the stimulation seen following an exposure with submaximal concentrations of PTH for as little as 2 minutes. Preincubation with the 25-OH-D₃ or 1,25-(OH)₂-D₃ resulted in a time dependent decrease in the enhancement of PTH response over a 1 hr period. It is, therefore, suggested that cyclic AMP may be involved in some aspects of the action of vitamin D₃ derivatives on bone cells.     

Comparative studies on the contents of vitamin D3, 25-hydroxy vitamin D3 and 7-dehydrocholesterol in fish liver

• 1.1. The contents of vitamin D₃, 25-hydroxyvitamin D₃ (25-OH-D₃) and 7-dehydrocholesterol (7-DHC) in 22 kinds of fish liver samples were determined by a high-performance liquid chromatographic (HPLC) method.
• 2.2. Vitamin D₃ was detected in all fish liver samples, but its contents varied from 84 to 264,000 ng/g wet tissue. The liver of fish belonging to Carangidae and Scombridae contained large amounts of the vitamin and therefore we deduced that vitamin D₃ levels in liver might have some relations with taxonomical positions of fishes.
• 3.3. 25-OH-D₃ was detected in 7 out of 22 kinds of fish liver samples, while 7-DHC was in 14 out of 22. The contents of the two sterols were generally much lower than those of vitamin D₃ and there was no special relationship between the contents of the sterols and the vitamin.

     

Metal ion-biomolecule interactions. III. Versatility of metal ion binding to imidazoles. Synthesis and 1H nmr spectroscopic properties of N- and C-bound mercury(II) complexes

Methylmercury(II) and mercury(II) complexes of imidazole (1), 1-methylimidazole (2), and the 1,3-dimethylimidazolium ion (3) have been prepared in aqueous or ethanolic solution. Elemental analysis and ¹H nmr spectroscopy have been used to characterize the complexes. The MeHg (Me = methyl) binding sites have been identified as N₁, N₃ (1), N₃, C₂ (2), and C₂ (3). Reaction with HgO leads to the formation of Hg-bridged complexes of the type Im-Hg-Im, (Im = imidazole), where bonding occurs through N₁ (1) and C₂ (3); the latter is also formed as a result of symmetrization of the C₂-bound MeHg complex. The formation of the C₂-bound (carbene) complexes is discussed in terms of the increased acidity of the C₂ proton resulting from coordination of an electrophilic species at N₃. Based on electrostatic considerations, there appears to be a “minimum degree of activation” required before C₂ bonding can occur, which explains the lack of this coordination mode in 1. ¹⁹⁹Hg-¹H spin-spin coupling (⁴J) is observed for C-bound mercury, but not for N-bound mercury, which is interpreted in terms of a decreased ligand exchange rate in the former case, due to the greater stability of the Hg-C bond. ²J coupling constants measured in (CD₃)₂SO for a number of MeHg complexes of heterocyclic ligands (including the imidazoles of the present study) correlate well with the ligand pK_a (25°C, aqueous solution), according to ²J = ?3.88 pK_a + 248.5. Results in the present work are discussed in relation to our previous work with nucleosides. The significance of the results to biological systems is considered.     

A new cycloartane-type triterpene and a new eicosanoic acid ester from fruits of Paullinia pinnata L.

A phytochemical study of the MeOH-soluble portion from the CH₂Cl₂/MeOH extract of the fruits of Paullinia pinnata resulted in the isolation of a new triterpenoid, cyclopinnatol (1), and a new eicosanoic acid ester, paulliniester (2), together with five known compounds including cycloart-22(E)-ene-3β,25-diol (3), cycloartenol (4), β-sitosterol (5), betulonic acid (6) and oleanonic acid (7). The structures of the new compounds were elucidated by spectroscopic analyses (NMR and MS) and comparisons with published data. Cyclopinnatol (1) and the MeOH-soluble portion exhibited significant and weak antibacterial activities against Staphylococcus aureus, with MICs of 32 and 50 μg/mL, respectively.     

Microbial transformation of the triterpene nigranoic acid in Trichoderma sp.

Two new metabolites were obtained by microbial transformation of the triterpene nigranoic acid (3,4-secocyloarta-4 (28), 24 (Z)-diene-3,26-dioic acid), (1) in the culture of Trichoderma sp. JY-1, a fungus obtained from the branches of Kadsura angustifolia. Their structures were established as 15α, 16α-dihydroxy-3,4-secocyloarta-4 (28), 17 (20), 17 (E), 24 (E)-triene-3,26-dioic acid (2) and 16α, 20α-dihydroxy-18 (13 → 17β) abeo-3,4-secocyloarta-4 (28), 12 (13), 24 (Z)-triene-3,26-dioic acid (3) by analysis of NMR and MS data and by analogy with the data for the substrate nigranoic acid (1). Compound 2 was found to possess an unusual 17(20), 17 (E)-ene structure while compound 3 featured an unprecedented 18(13 → 17β)-abeo-secocyloarta skeleton. Additionally, compounds 1–3 showed weak anti-HIV activity with EC₅₀ values of 10.5, 8.8 and 7.6 μg/mL, therapeutic index values (CC₅₀/EC₅₀) of 8.48, 9.12 and 10.1, respectively.     

Isolation and identification of 23,25-dihydroxy-24-oxo-vitamin D3: A metabolite of vitamin D3

A new metabolite of vitamin D₃ has been isolated in pure form from incubations of rat kidney homogenates with 25-hydroxyvitamin D₃ [25-OH-D₃]. It was identified as 23,25-dihydroxy-24-oxo-vitamin D₃ [23,25(OH)₂-24-oxo-D₃] by means of ultraviolet absorption spectrophotometry and mass spectrometry. Also, 25-OH-D₃-26,23-lactone and 24R,25-dihydroxyvitamin D₃ were obtained from the same incubation mixtures. The enzyme activity responsible for the conversion of 25-OH-D₃ to 23,25(OH)₂-24-oxo-D₃ was induced by perfusion of the kidneys $\text{in}\text{vitro}$ with 50 nM 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃].     

The mechanism of acid-catalyzed conversion of ginsenoside Rf and two new 25-hydroxylated ginsenosides

Ginsenoside Rf is known to have higher chemical stability than other ginsenosides and until lately, the constituents in which it would convert were not known. Only in recent times, it was found that ginsenoside Rf converted to (20E)-Rg₉, (20Z)-Rg₉, Rg₁₀, and 20(R)-Rf. During my continued studies to update the chemical profile of red ginseng, two new ginsenosides converted from ginsenoside Rf, 25-hydroxylated ginsenosides, were discovered. These two new converted ginsenosides, namely (20E),25(OH)-ginsenoside Rg₉ (1), and (20Z),25(OH)-ginsenoside Rg₉ (2), together with ginsenosides (20E)-Rg₉ (3), (20Z)-Rg₉ (4), Rg₁₀ (5), and 20(R)-Rf (6) were isolated from a reaction mixture of ginsenoside Rf in an acid-catalyzed reaction. Their chemical structures (1 and 2) were elucidated by NMR and Mass spectral methods. Compounds 1 and 2 were presumably generated by hydration of (20E)-, and (20Z)-ginsenoside Rg₉. The presence of these six converted ginsenosides was confirmed by UPLC/TOF-MS method in red ginseng. On the basis of these results, I deduced the overall conversion mechanism of ginsenoside Rf and evaluated the significance of ginsenoside Rf as a characteristic mark substance of Panax ginseng.     

Cytotoxic triterpenoid saponins from Aesculus glabra Willd.

Twenty-four acylated polyhydroxyoleanene saponins were isolated from the seeds of Aesculus glabra. Sixteen of them, namely aesculiosides G1–G16 (1–16), were determined as compounds by spectroscopic and chemical analysis. The structural features of all 24 saponins are: (1) arabinofuranosyl units affixed to C-3 of the glucuronopyranosyl unit in the trisaccharide chain; (2) no 24-OH substitution; (3) C-2 sugar moiety substitution of the 3-O-glucuronopyranosyl unit is either glucopyranosyl or galactopyranosyl. The features of these isolated saponin structures provide more evidence for chemical taxonomy within the genus Aesculus. The cytotoxicity of the aesculiosides (1–16) were tested against A549 and PC-3 cancer cell lines with GI₅₀ from 5.4 to >25 μM.     

Calmodulin inhibitors from the fungus Emericella sp.

Two new xanthones identified as 15-chlorotajixanthone hydrate (1) and 14-methoxytajixanthone (2) were isolated from an Emericella sp. strain 25379 along with shamixanthone (3) and tajixanthone hydrate (4). The stereostructures of 1 and 2 were elucidated by spectroscopic and molecular modeling methods. The absolute configuration at the stereogenic centers of 1 was established according to CD measurements. In the case of 2, however, the absolute configuration at C-20 and C-25 was designated as S and R, respectively, by Mosher ester methodology. Thereafter, the configuration at C-14 and C-15 of 2 was established as S and S, respectively by comparing the optical rotation and ¹H–¹H coupling constant experimental values with those obtained through molecular modeling calculations at DFT B3LYP/DGDZVP level of theory for diasteroisomers 2a–2d. The activation of the calmodulin-sensitive cAMP phosphodiesterase (PDE1) was inhibited in the presence of 1–4 in a concentration-dependent manner. The effect of compounds 2 (IC₅₀ = 5.54 μM) and 4 (IC₅₀ = 5.62 μM) was comparable with that of chlorpromazine (CPZ; IC₅₀ = 7.26 μM), a well known CaM inhibitor used as a positive control. The inhibition mechanism of both compounds was competitive with respect to CaM according to a kinetic study. A docking analysis with 2 and 4 using the AutoDock 4.0 program revealed that they interacted with CaM in the same pocket as trifluoropiperazine (TFP).     

Two new polycyclic polyprenylated acylphloroglucinols from Hypericum curvisepalum N. Robson

Phytochemical investigation of the whole plant of Hypericum curvisepalum led to the isolation of nine polycyclic polyprenylated acylphloroglucinols (1–9). Among them, curvisepalumiones A (1) and B (2) are two new compounds and their structures were elucidated based on extensive spectroscopic data analysis combined with Rh₂(OCOCF₃)₄-induced electronic circular dichroism experiment. Cytotoxic assay showed that all the tested compounds except for 1 was moderately cytotoxic against human hepatocarcinoma cell line SMMC-7721 cell line, while compounds 3 and 4 also exhibited cytotoxicity toward MGC-803 cells. In addition, compound 2 exhibited weak inhibitory activity against Bacillus subtilis.     

Alkynyl cobalt complexes. An electrochemical study

The preparation and characterisation of the complexes Co₂(CO)₅(PMe₃)(μ-η²-Me₃SiC₂CCSiMe₃) (2) and Co₂(CO)₄(PMe₃)₂(μ-η²-Me₃SiC₂CCSiMe₃) (3) are described. A comparative electrochemical study of the complexes Co₂(CO)_6−nL_n(μ-η²-Me₃SiC₂CCSiMe₃) (n=0 (1); n=1, L=PMe₃ (2); n=2, L=PMe₃ (3), PPh₂Me (4), dppa (5), dppm (6)) is presented by means of the cyclic and square-wave voltammetry techniques. Substitution of CO by phosphine ligands transforms the Co₂C₂ redox centre from a readily reducible to an easily oxidisable centre and contributes to the stabilisation of the Co–Co bond increasing the lifetime of the radical cations and anions.