High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay

Background

Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment.

Methods

A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP^® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals.

Results

The average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R² = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24).

Conclusions

High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.     

Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b₅ and NADH-cytochrome b₅ reductase from the microsomal fraction of Pisum sativum. The cytochrome b₅ component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b₅ component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.     

A change of osteocalcin (OC) and tartrate resistant acid phosphatase 5b (TRACP-5b) with the menstrual cycle

Bone metabolism markers associated with 4 menstrual cycle phases were evaluated in 14 healthy young females without menstrual disorder. Menstrual cycle phases were confirmed with basal body temperature for 3 months, luteinizing hormone kits, and sexual hormone concentrations of serum. The bone metabolism markers used were osteocalcin (OC), which was measured by immunoradiometric assay (IRMA), and tartrate resistant acid phosphatase 5b (TRACP-5b), which was measured by enzyme immunometric assay (EIA). The highest values of OC and TRACP-5b were observed in the ovulation phase, and TRACP-5b increased significantly when compared with levels in the menstrual phase (p<0.05). Furthermore, the changes in sex-hormone secretion involved in OC and TRACP-5b showed specific patterns during the menstrual cycle. In other words, TRACP-5b levels are influenced by sex hormones produced during the menstrual period and are based on the bone-formation status. Therefore, it is presumed that the TRACP-5b levels during ovulation play a central role in bone formation and bone metabolism.     

Optically active transition-metal compounds. 93. X-ray determination of the structure and absolute configuration of (+) 578-C5H5Fe(CO) [P(C6H5)3] COCH3

The structure and absolute configuration of (+)₅₇₈- C₅H₅Fe(CO)[P(C₆H₅)₃]COCH₃ have been determined by single crystal X-ray diffraction methods. The substance crystallizes in the monoclinic space group P2₁ with cell constants of a = 8.084(14), b = 8.527(2), c32.706(21) Å and β = 104.32(10)°; V 2184.18 ų and D(calc: Z 4 mol/unit cell) = 1.381 g cm^-3. There are two independent molecules in the asymmetric unit, which allowed us to gauge the effect of packing on the conformation of those groups able, in principle, to be twisted by crystalline forces. Only minor changes in conformations were observed, the largest being at the terminal CH₃ of the acetyl ligand (0.065 Å). All other differences in conformation are less than 0.036 Å. The plane of the acetyl ligand is close to being aligned with the FeC(CO) bond, making the acetyl oxygen point in the direction of the phosphorus atom. It is suggested that in phosphine exchange reactions this conformation persists in solution while the acetyl oxygen, intra- molecularly, attacks the adjacent phosphorus atom to form a dihapto acetyl species as the first intermediate, in which there is retention of configuration at Fe.With the priority of the ligand sequence as C₅H₅ > P(C₆H₅)₃ > CO > COCH₃, the absolute configuration at Fe is (S). So, the formation of (−)₅₇₈- C₅H₅Fe(CO)[P(C₆H₅)₃]COCH₃ by reaction of (+)₅₇₈- C₅ H₅ Fe(CO)[P(C₆H₅)₃] COOC₁₀H₁₉ and LICH₃ requires an inversion to occur at the Fe center.     

Allergen-specific responses of CD19(+)CD5(+)Foxp3(+) regulatory B cells (Bregs) and CD4(+)Foxp3(+) regulatory T cell (Tregs) in immune tolerance of cow milk allergy of late eczematous reactions

Foxp3-expressing cells among CD19(+)CD5(+) B cells were identified as regulatory B cells. Food allergy manifesting as late eczematous reactions is regarded as a non-IgE-mediated food allergy. The diagnosis for milk allergy manifesting as late eczematous reactions was made on the basis of the findings obtained from a double-blind placebo-controlled food challenge in patients with atopic dermatitis. Twelve patients with milk allergy and 12 patients who could tolerate milk were selected. On casein stimulation, the CD19(+)CD5(+)Foxp3(+) B cell (Breg) fraction in CD5(+) B cells decreased from 4.4±1.1% to 3.1±0.7% (P=0.047, n=12) in the milk allergy group and increased from 4.4±1.3% to 5.2±1.4% (P=0.001, n=10) in the milk-tolerant group. On the other hand, on allergen stimulation, the number of CD4(+)Foxp3(+) regulatory T cells (Tregs) in the milk allergy group and milk-tolerant group increased from 2.6±0.7% to 3.4±0.6% (P=0.014, n=9) and from 2.7±1.0% to 3.5±1.0% (P=0.038, n=10), respectively. In conclusion, allergen-specific responses of Bregs, rather than those of Tregs, seem to influence the immune responses (i.e., allergy or tolerance) to a food allergen.     

The efficiency of the action of alpha-tocopherol and its homologs on luminol-dependent chemiluminescence induced by (Fe2+ + NADP.H) and (Fe2+ + ascorbate) systems in rat liver microsomes]

The effects of alpha-tocopherol (C16) and its homologues with different chain length (6-hydroxychromanes-C1, C6, C11) on lipid peroxidation induced luminol-dependent chemiluminescence in rat liver microsomal suspensions were studied. It was shown that C1, C6 and C11 inhibited the (Fe(2+) + ascorbate)-and (Fe(2+) + NADP.H)-induced chemiluminescence. The inhibitory effect was decreased in the order: C1 C6 C11, C16 was not influenced chemiluminescence. The possible reason underlying these differences was discussed: different efficiency of interaction of C16 and its homologues with hydroxyl and superoxide radicals, which initiate the luminol-dependent chemiluminescence. It was concluded that C16 (in concentration below 0.5 mM) was not interacted with hydroxyl and superoxide free radicals, generated in microsomal suspensions under (Fe(2+) + ascorbate)- and (Fe(2+) + NADP.H)-dependent lipid peroxidation.     

Enantiospecific synthesis of carbocyclic (+)-(E)-5-(2-bromovinyl)-2'-deoxyuridine

(+)-1-[(1R, 3S, 4R)-3-hydroxy-4-hydroxymethylcyclopentyl]-5-[(E)-2- bromovinyl]-1H,3H-pyrimidin-2,4-dione 10 was synthesized starting from (+)-endo-5-norbornen-2-yl acetate. This chiral educt was obtained by enzymatic hydrolysis of racemic esters of endo-5-norbornen-2-ol.     

Interaction of 5-iminodaunorubicin with Fe(III) and with cardiolipin-containing vesicles

5-Iminodaunorubicin is an anthracycline derivative exhibiting promising antitumor activity. Using potentiometric and spectroscopic measurements we have shown that 5-iminodaunorubicin forms with Fe(III) a complex in which three molecules of drug are bound to one Fe(III) ion. Each molecule is chelated through the C-12-carbonyl and the C-11-phenolate oxygen atoms. The stability constant is 1.6 X 10(34). Using circular dichroism measurements we have studied the interactions of 5-iminodaunorubicin with cardiolipin-containing vesicles. We have shown that cardiolipin could bind one molecule of drug without penetration of the dihydroanthraquinone moiety into the bilayer.     

Cytochrome b(5) forms homomeric complexes in living cells

Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis.     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

Carbon monoxide complex of cytochrome b(5) at acidic pH

CO complex of cyt b(5) generated at acidic pH is investigated by absorption, resonance Raman (RR), and far UV CD measurements. The Soret maximum wavelength blue-shifted to 420 nm with other absorption bands observed around 540 and 570 nm for reduced cyt b(5) upon interaction with CO at acidic pH (pH 3.1-3.5). Under this condition, the iron-carbon stretching RR band was observed at 529 cm(-1) (520 cm(-1) for C(18)O), which indicated formation of a heme&bond;CO adduct with a histidine as an axial ligand. Heme dissociated from the reduced cyt b(5) protein at pH approximately 3.5, whereas its rate decreased under CO atmosphere compared with N(2) atmosphere, due to formation of a heme&bond;CO adduct with a histidine as an axial ligand.     

Facilitation of Fe(II) antoxidation by Fe(III) complexing agents

The rate of oxidation of Fe(II) by atmospheric oxygen at pH 7.0 is significantly enhanced by low molecular weight Fe(III)-complexing agents in the order EDTA ≈ nitrilotriacetate > citrate > phosphate > oxalate. This simple effect of Fe(III) binding probably accounts for the “ferroxidase” activity exhibited by transferrin and ferritin.     

Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5)

A soluble cytochrome b(558) from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b(5) homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue.     

Molecular dynamics simulation of the DNA triplex d(TC)5.d(GA)5.d(C+T)5.

A molecular dynamics simulation of the DNA triple helix d(TC)5.d(GA)5.d(C+T)5 is described (C+ represents a protonated cytosine residue). The simulation has been performed using the program AMBER 3.1 and includes counterions and explicit solvent under periodic boundary conditions. Both the dynamic and time-averaged behaviour of the system has been analysed. Considerable deviations from the fibre-diffraction model for DNA triple helix structure are observed, including the repuckering of the purine strand sugars that has been identified in some nuclear magnetic resonance (n.m.r.) studies. The simulation suggests that this conformational change may be driven by the possibility of improved interactions between the phosphate groups of this strand and both the solvent and counterions. Several examples of a particular conformational transition are observed, involving correlated changes in the backbone angles alpha and gamma. These transitions provide a possible explanation for some unusual n.m.r. data that have been reported. The structure of the triple helix major groove also suggests an explanation for the observed stabilization of DNA triplexes by polyvalent cations, and their ability to interact with drugs that bind in the minor groove of DNA duplexes.