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PubMed2001

Increasing uptake and bioactivation with development positively modulate diazinon toxicity in early life stage medaka (Oryzias latipes).

Diazinon, an organophosphate pesticide, becomes biotransformed to a more potent oxon metabolite that inhibits acetylcholinesterase (AChE). Early life stages (els) of medaka, Oryzias latipes, were used to determine how development of this teleost affects sensitivity to diazinon. With developmental progression, from day of fertilization to 7-day-old larvae, we found that the 96-h LC50 and AChE IC50 values decreased, indicating greater host sensitivity to diazinon upon continued development. We then examined changes in AChE activity, its inhibition by the active metabolite diazoxon, and uptake and bioactivation of the compound. AChE activity remained low during much of development but increased rapidly just prior to hatch. In addition, in vitro incubation of tissue homogenates from embryos or larvae showed no differences in the sensitivity of AChE to diazoxon. Uptake studies with 14C-diazinon revealed greater body burdens of 14C as medaka developed. In addition, AChE IC50 values determined by in vivo exposure to diazoxon were greater in larvae than in embryos. Because diazinon is bioactivated by the P450 enzyme system, two P450 inhibitors were used in vivo to explore the role of metabolism in sensitivity. When exposure to diazinon occurred in the presence of increasing amounts of piperonyl butoxide (PBO), AChE inhibition decreased in a dose-response fashion and 2.0 x 10(-5) M PBO alleviated any difference in inhibition between larvae and embryos. However, PBO did not alter total 14C uptake when exposed simultaneously with 14C-diazinon, nor did it affect AChE inhibition using diazoxon. Controls ruled out differential effects of PBO on uptake and inhibition. In addition, a second general P450 inhibitor, 1-aminobenzotriazole, also decreased AChE inhibition. Finally, using exogenous acetylcholinesterase as a trap for the oxon metabolite, larval microsomes displayed greater bioactivation of diazinon than did a microsomal preparation from embryos. Taken together, results suggest that uptake and bioactivation are working to enhance diazinon sensitivity in this developmental model of a teleost fish.

Acetylcholinesterase↗

PubMed1997

Follicle-stimulating hormone stimulates circulating gonadotropin surge-attenuating/inhibiting factor bioactivity in cows.

This study aimed to determine whether superovulation in cattle stimulates gonadotropin surge-attenuating/inhibiting factor (GnSAF/IF) bioactivity, as it does in humans. Blood samples were collected from cows (n = 7 per treatment) at -4, 8, 20, 32, 44, 56, and 68 h after injections of saline, eCG, or FSH. Equal volumes of plasma at each treatment and time point were pooled, and GnSAF/IF and inhibin bioactivities were measured using an established rat pituitary cell culture bioassay. Plasma from saline- and eCG-treated cows had little effect on GnRH-induced LH secretion (116.3 +/- 8.3%-81.6 +/- 6.0% of control), while plasma from FSH-treated cows produced a time-dependent suppression of GnRH-induced LH secretion, falling to 64.6 +/- 4.0% of the control value at 56 h after first FSH injection (p < 0.001). The GnSAF/IF bioactivity from the 56-h plasma eluted at pH 5.73 by pseudochromatofocusing-similar to the GnSAF/IF isoelectric point value of 5.81 determined using serum from superovulated women. Plasma from FSH-treated cows reduced basal FSH secretion more than plasma from eCG-treated cows (to 55.5 +/- 5.7% and 63.2 +/- 6.6% of the control value, respectively, p < 0.01) although immunoreactive inhibin concentrations were similar between the two groups. We conclude that FSH, but not eCG, treatment causes a time-dependent production of circulating GnSAF/IF bioactivity in cattle.

Animals↗

PubMed1998

Ovine allantoic fluid contains high concentrations of activin A: partial dissociation of immunoactivity and bioactivity.

In a preliminary study, allantoic fluid collected from pregnant sheep across gestational ages of 20-124 days contained significantly higher levels of activin bioactivity (189 +/- 74 ng/ml, mean +/- SE) than did amniotic fluid (3.2 +/- 0.6 ng/ml). Using a combination of chromatography steps, we isolated from 5 L of allantoic fluid approximately 612 microg of immunoactive activin, which eluted over 10 fractions from a C8 reversed-phase column. When these fractions were assayed in a rat pituitary cell culture bioassay, in a specific RIA, and in an activin A two-site ELISA, the RIA activity was skewed to the less hydrophobic side of the activin profile, while the bioactivity was skewed to the more hydrophobic forms. The activity measured in the two-site ELISA more closely matched the mass of activin as determined by laser densitometry. Amino-terminal sequencing of fractions containing either peak immunoactivity or bioactivity showed each to be identical to activin A. This was confirmed by internal sequences from a fraction that eluted in the area of overlapping immunoactivity and bioactivity. A peptide containing at least 18 amino acids at its amino terminus, which were identical to the conserved region of the acute-phase protein serum amyloid A, was identified in the most immunoactive activin fractions.

Activins↗

PubMed1993

Bioactive glass particles of narrow size range: a new material for the repair of bone defects.

A clinical trial to treat dental osseous lesions with bioactive glass granules of narrow size range (300 to 360 microns) has been conducted since early spring 1990. This study followed an animal study in which the osseous tissue repair properties of bioactive glass granules of narrow size range and hydroxyapatite granules were compared for up to 2 years of implantation. The therapeutic response to the bioactive glass material exceeded the response to the hydroxyapatite as evidenced by very extensive osteoconduction, as well as the capacity to cause differentiation of osteoprogenitor cells to osteoblasts. The clinical study was started by virtue of the bioactive glass granules of narrow size range eliciting expeditious bone tissue formation throughout a defect. In this clinical study 87 patients and 106 defects were treated. The indications selected were apical resection areas, cystic defects, extraction sites, and defects of the alveolar ridge due to surgery or resorption. After insertion, the particles remained well in place and only small changes in the contours of the restored defects were seen, at the most up to 2 months postinsertion. At 3 months the application sites had fully solidified. Radiographic analysis indicated that the material integrated into the bone tissue, and at 6 months any difference between glass particles and bone tissue had nearly disappeared. The few initial cases with limited clinical results were caused by factors unrelated to the glass granules, mostly the surgical technique. By adapting the surgical technique, no unfavorable clinical results were subsequently experienced.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed2005

Vertebroplasty by use of a strontium-containing bioactive bone cement.

STUDY DESIGN: A review of the laboratory and clinical data for a new strontium-containing hydroxyapatite bioactive bone cement. OBJECTIVES: To compare the properties of the strontium-containing bioactive bone cement with those of polymethyl methacrylate (PMMA) and hydroxyapatite (HA) bone cements. SUMMARY OF BACKGROUND DATA: Vertebroplasty and kyphoplasty using conventional PMMA bone cements have been effectively used to treat osteoporotic spine fractures with good short- and medium-term results. However, PMMA has some undesirable properties, including its high setting temperature, lack of osseointegration, and large stiffness mismatch with osteoporotic bone. These properties are responsible for some postoperative complications. METHODS: Strontium-containing hydroxyapatite (Sr-HA) bioactive bone cement consists of a filler blend of strontium-containing hydroxyapatite, fumed silica and benzoyl peroxide; and a resin blend of bisphenol A diglycidylether methacrylate, triethylene glycol dimethacrylate, poly(ethylene glycol) methacrylate, and N, N-dimethyl-p-toluidine. Its properties, including mechanical strength, setting temperature, biocompatibility, and osseoinduction, were compared with other cements in vitro and in vivo. Early clinical results are presented. RESULTS: The Sr-HA cement has a setting time of 15 to 18 minutes, a maximum setting temperature of 58 degrees C, a compressive strength of 40.9 MPa, bending strength of 31.3 MPa, and a bending modulus of 1,408 MPa. The bending strength and modulus are closer to human cancellous bone. Sr-HA cement promotes osteoblast attachment and mineralization in vitro and bone growth and osseointegration in vivo. In a pilot study, 23 cases of osteoporotic fractures treated with this cement with a mean follow-up of 18 months suggest that it is as effective as PMMA in relieving pain. DISCUSSIONS: Oral strontium has been shown to induce new bone formation and is effective in reducing fracture risk in osteoporosis. Our data suggest that strontium delivered locally has the same effect; thus, the combination of strontium with HA in a cement with a low setting temperature, adequate stiffness, and low viscosity makes this a good bioactive cement for vertebroplasty and kyphoplasty.

Animals↗

PubMed1985

On the stability in vitro of bioactive human adrenocorticotrophin in blood and plasma.

The disappearance in vitro of ACTH from whole human blood and plasma held at 22 degrees C has been monitored using a dispersed adrenal cell bioassay and an unextracted radioimmunoassay. In three normal subjects after a metyrapone test and two patients with Addison's disease, endogenous bioactive ACTH levels were unchanged for at least 1 h in blood and 2 h in plasma. Moreover greater than 50% of the bioactive plasma ACTH was still present in the plasma samples from the patients with Addison's disease after 24 h incubation at ambient temperatures. Human pituitary ACTH (1-39), spiked into plasma from dexamethasone suppressed subjects to give a concentration of 250 ng/l, was stable by bioassay for at least 2 h. No loss of biological activity was observed on subjecting plasma from a patient with Addison's disease or spiked plasma to two cycles of thawing at 37 degrees C and freezing at -70 degrees C or thawing at 20 degrees C and freezing at -20 degrees C. Some loss of bioactivity (20%) occurred on subjecting the patient's, but not ACTH-spiked plasma to four cycles of thawing at 20 degrees C/freezing at -20 degrees C. We conclude that bioactive ACTH (endogenous or exogenous) may be more stable in vitro in human blood and plasma than has been previously thought. If our studies can be confirmed in a larger series then it may be that conditions for handling blood specimens for ACTH assays could be reappraised.

Addison Disease↗

PubMed1994

Effects of oestrogen treatment on serum gonadotrophin bioactivity, immunoreactivity and isohormone distribution, and on immunoreactive inhibin levels, in prostatic cancer patients.

OBJECTIVE AND DESIGN: No data are available on effects of long-term exposure to oestrogen on bioactivity of gonadotrophins in men. We studied the effects of a 6-month oestrogen therapy on serum FSH and LH bioactivity (B), immunoreactivity (I) and isohormone distribution, and on serum I-inhibin levels, in patients with prostatic carcinoma. PATIENTS: Eleven men with advanced prostatic cancer were studied, each receiving 160 mg of polyoestradiol phosphate (Estradurin) once a month intramuscularly for 6 months. MEASUREMENTS: Serum samples were collected before, and after 2 and 6 months of oestrogen treatment. Serum B- and I-FSH levels were measured by immature rat granulosa cell bioassay and immunofluorometric (IFMA, Delfia) assay, respectively, and those of B- and I-LH by mouse interstitial cell bioassay and IFMA, respectively. Serum oestradiol (E2) concentrations were measured by IFMA assay, and serum testosterone (T) and inhibin levels by radioimmunoassay. Isoelectric focusing was used for fractionation of the FSH and LH isoforms. RESULTS: The pretreatment levels of B-FSH and I-FSH were 84.7 +/- 21.6 and 11.4 +/- 3.2 IU/l (mean +/- SEM), respectively, and the B/I ratio of FSH was 8.3 +/- 1.0. The pretreatment levels of B-LH and I-LH were 23.5 +/- 3.2 and 10.1 +/- 2.3 IU/l, respectively, and the B/I ratio was 3.0 +/- 0.4. After 6 months of oestrogen therapy, B-FSH and I-FSH decreased to 37.5 +/- 8.1 (P < 0.05) and 1.3 +/- 0.3 IU/l (P < 0.01), respectively, but the B/I ratio of FSH increased to 28.5 +/- 4.2 (P < 0.05). B- and I-LH levels decreased in 6 months to 7.4 +/- 0.9 and 2.3 +/- 0.5 IU/l (P < 0.01), respectively, but no change was found in the B/I ratio of LH. Serum T levels decreased from 19.0 +/- 2.6 to 2.7 +/- 0.9 nmol/l (P < 0.01) during the 6-month treatment, and the respective E2 levels increased from 0.2 +/- 0.01 to 4.4 +/- 0.5 nmol/l (P < 0.01). Serum I-inhibin levels were analysed from eight patients. The levels at 0, 2 and 6 months were 0.81 +/- 0.09, 0.50 +/- 0.03 and 0.54 +/- 0.01 microgram/l, respectively. Gonadotrophins in the pretreatment and 6-month samples of four patients were analysed by isoelectric focusing. In FSH of all subjects, and in LH of three subjects, a shift from acidic to more basic isoforms occurred after oestrogen therapy. This is in keeping with the increase of the B/I ratio of FSH. With LH, the isoform shift occurred between fractions with similar B/I ratios, and hence there was no shift in the overall B/I ratio. CONCLUSIONS: Oestrogen therapy of men suppressed bioactive and immunoreactive levels of gonadotrophins. The B/I ratio of FSH increased, and this increase was associated with a shift in the isohormone profile to more basic forms. In contrast, no change occurred in the B/I ratio of LH, even though changes in the isohormone profile were observed. Hence, not all changes in the isohormone distribution of gonadotrophins result in changes of the intrinsic in-vitro bioactivity.

Aged↗

PubMed1995

Human endothelial cells bioactivate organic nitrates to nitric oxide: implications for the reinforcement of endothelial defence mechanisms.

Although in therapeutic use for more than a century, the mode of cellular action of organic nitrates remains incompletely understood. Despite ample experimental evidence from animal studies to show that nitrates are metabolized to NO in the vascular smooth muscle, direct demonstration of such an activity in human vascular cells is still lacking. Moreover, the role of the endothelium in modulating the pharmacodynamic action of nitrates is far from clear. We therefore aimed to investigate whether or not human endothelial cells are capable of bioactivating these drugs to NO and whether the amounts generated are sufficient to elicit any biological effects. Using cultured human umbilical vein endothelial cells (HUVECs) as an established model system a combination of three different methods was used to address this issue: (1) quantification of NO formation upon endothelial nitrate metabolism using the oxyhaemoglobin technique; (2) evaluation of the second messenger response using radioimmunoassay for cGMP; and (3) assessment of mechanism and extent of potentiation of the anti-aggregatory effect of nitrates in the presence of endothelial cells as a relevant bioassay. We now show that superfusion of cultured human endothelial cells on microcarrier beads with either glyceryl trinitrate (GTN) or isosorbide dinitrate (ISDN; both at 0.1-100 mumol L-1) results in a concentration-dependent formation of NO. NO generation from isosorbide 5-mononitrate (IS-5-N) was below the detection limit. The amounts of NO produced (maximally 2.97 +/- 0.98 pmoles NO min-1 x mg protein with 100 mumol L-1 GTN; n = 8) were similar to those elicited upon challenge of the cells with 100 nM bradykinin. NO formation from either organic nitrate was accompanied, in a concentration-dependent and methylene blue-inhibitable manner, by stimulation of endothelial soluble guanylyl cyclase with consequent increases in the intracellular level of cGMP (maximally 32-fold over basal levels with ISDN), a significant portion of which was released into the extracellular space. Upon continuous 30 min superfusion or repeated application of high concentrations of GTN (100 mumol L-1) nitrate bioactivation to NO was subject to partial tachyphylaxis. Co-incubation of washed human platelets with HUVECs potentiated the anti-aggregatory action of nitrates in a cell number dependent and oxyhaemoglobin-sensitive manner and this effect, too, was accompanied by increases in intraplatelet cGMP levels. The potentiating effect was largely inhibited after blockade of sulfhydryl groups by pre-incubation of HUVECs with N-ethylmaleimide and completely abrogated after pretreatment of cells with the tissue fixative glutaraldehyde. These results demonstrate that human endothelial cells are capable of bioactivating organic nitrates to NO by an enzymatic, apparently thiol-sensitive pathway, in quantities sufficient to influence endothelial and platelet function. Besides the well known vasorelaxant action of organic nitrates, which is mainly due to their metabolism in the smooth muscle compartment, these drugs may therefore be endowed with a hitherto underestimated potential to directly influence endothelial functions via the NO/cGMP pathway. Through specific bioactivation in the endothelium itself organic nitrates can thus mimic and reinforce protective functions normally served by a functional endothelium such as the modulation of blood cell/vessel wall interactions and inhibition of cell proliferation.

Biotransformation↗

PubMed2004

Bioactivation of 1,1-dichloroethylene by CYP2E1 and CYP2F2 in murine lung.

1,1-Dichloroethylene (DCE) exposure evokes lung toxicity with selective damage to bronchiolar Clara cells. Recent in vitro studies have implicated CYP2E1 and CYP2F2 in the bioactivation of DCE to 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. An objective of this study was to test the hypothesis that bioactivation of DCE is catalyzed by both CYP2E1 and CYP2F2 in murine lung. Western blot analysis of lung microsomal proteins from DCE-treated CD-1 mice showed time-dependent loss of immunodetectable CYP2F2 and CYP2E1 protein. Dose-dependent formation of conjugate [C] was observed in the lungs of CD-1 mice treated with DCE (75-225 mg/kg), but it was not detected after pretreatment with 5-phenyl-1-pentyne (5-PIP). Treatment of mice with 5-PIP and also with diallyl sulfone (DASO2) significantly inhibited hydroxylation of p-nitrophenol (PNP) and chlorzoxazone (CHZX). Incubation of recombinant CYP2F3 (a surrogate for CYP2F2) and recombinant CYP2E1 with PNP and CHZX confirmed that they are substrates for both of the recombinant enzymes. Incubation of the recombinant enzymes with DASO2 or 5-PIP significantly inhibited hydroxylation of both PNP and CHZX. Bronchiolar injury was elicited in CD-1 mice treated with DCE (75 mg/kg), but it was abrogated with 5-PIP pretreatment. Bronchiolar toxicity also was manifested in the lungs of CYP2E1-null and wild-type mice treated with DCE (75 mg/kg), but protection ensued after pretreatment with 5-PIP or DASO2. These results showed that bioactivation of DCE in murine lung occurred via the catalytic activities of both CYP2E1 and CYP2F2 and that bioactivation by these enzymes mediated the lung toxicity.

Animals↗

PubMed1998

Nitric oxide inhibits vascular bioactivation of glyceryl trinitrate: a novel mechanism to explain preferential venodilation of organic nitrates.

Organic nitrates undergo enzymatic metabolization in the vasculature to release the active compound nitric oxide (NO). The resulting preferential venodilation has been suggested to be related to the vascular bioactivation process of organic nitrates because sodium nitroprusside, which is bioactivated differently, is not venoselective. We sought to determine whether NO has an influence on vascular bioconversion of organic nitrates because endogenous endothelial production of NO is smaller in veins than in arteries. Rings of porcine coronary arteries were subjected to radioactive glyceryl trinitrate (GTN) after preincubation with defined amounts of NO. The vascular content of GTN and the dinitrates (GDNs) 1,2-GDN and 1,3-GDN then was quantified. NO (3 microM, 30 min) significantly impaired bioactivation of GTN as indicated by a 30-50% reduction in the accumulation of 1,2-GDN and 1,3-GDN, whereas unchanged GTN was increased. Incubation with NO also reduced the stimulated specific activity of soluble guanylate cyclase isolated from human platelets. Its specific activity was reduced from 2.6 +/- 0.2 to 2.1 +/- 0.13 nmol of cGMP/mg/min. Relaxation studies with rings of porcine coronary arteries showed that NO-induced inhibition of vascular GTN metabolism and cGMP accumulation decreased the vasodilator potency of GTN by 10-fold. Further experiments showed that the duration of NO treatment is more important for this effect than the concentration of NO. We suggest that NO can inhibit vascular bioactivation of organic nitrates and might slightly desensitize soluble guanylate cyclase. The preferential venodilation induced by organic nitrates might be the result of the comparably low production of endogenous NO in veins.

Animals↗

PubMed1989

Cholecystokinin in pig plasma: release of components devoid of a bioactive COOH-terminus.

Using radioimmunoassays specific for different sequences of cholecystokinin (CCK), we studied the intestinal release of CCK in pigs. After stimulation by intraduodenal infusion of HCl, plasma CCK concentrations, measured with an antiserum specific for the sulfated, bioactive sequence of CCK, increased from 1.4 +/- 0.7 to 42.7 +/- 11.7 pM in portal plasma and from 0.5 +/- 0.3 to 12.3 +/- 1.5 pM in peripheral plasma. The concentrations measured with an antiserum specific for the NH2-terminal sequence 5-10 of CCK-33 were considerably higher, increasing from 64 +/- 17 to 139 +/- 14 pM in portal plasma and from 69 +/- 7 to 102 +/- 9 pM in peripheral plasma. Chromatography suggested that the NH2-terminal immunoreactivity consisted of large CCK fragments devoid of the bioactive COOH-terminal octapeptide, i.e., desocta and/or desnona CCK-58, CCK-39, and CCK-33. The bioactive forms in both portal and peripheral plasma comprised CCK-58-, CCK-33-, CCK-22-, CCK-12-, and CCK-8-like forms. Generally, the CCK-22-like component predominanated, but although CCK-8 was more abundant than CCK-33 in portal plasma, these forms occurred in equal amounts in peripheral plasma. Large amounts of NH2-terminal immunoreactivity were also found in the venous effluent of the isolated perfused duodenum after stimulation with gastrin-releasing peptide; the venous perfusate contained mainly CCK-22- and CCK-8-like material, which together constituted greater than 80% of the bioactive CCK components. Both duodenal and jejunal mucosa contained components resembling NH2-terminal fragments as well as large amounts of intact CCK-58 and CCK-33.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

Bioactive suture: a novel immunotherapy for head and neck cancer.

PURPOSE: We have proposed to characterize the mechanism through which bioactive surgical sutures generate a T(H)1 immune response and to define the immune-stimulating half-life of the sutures. EXPERIMENTAL DESIGN: Bioactive sutures of interferon gamma (IFNgamma), interleukin 2 (IL-2), anti-CD3/CD28, anti-CD3/CD28 + IL-2, or anti-CD3/CD28 + IFNgamma sutures were used to stimulate lymphocytes from normal donors and from head and neck cancer patients in vitro over a 24-day period. Cell supernatants were analyzed by ELISA, and T cells were phenotyped to characterize the immune response generated. Intracellular cytokine staining was performed to measure the expansion of flu-specific T cells. Electromobility shift assay and supershift assay were used to measure the intranuclear DNA binding activity of nuclear factor kappaB and its p65 subunit in T cells activated by sutures in the presence and absence of a proteasome inhibitor, MG-132. RESULTS: Anti-CD3/CD28, anti-CD3/CD28 + IL-2, or anti-CD3/CD28 + IFNgamma generated a prolonged T(H)1 immune response for 18 days in vitro. Anti-CD3/CD28 expanded flu-specific T cells. Activated T cells demonstrated enhanced CD40 ligand (CD40L) expression within 72 hours of stimulation, which stimulated other cells to secrete IL-12. Stimulated T cells demonstrated increased intranuclear expression of nuclear factor-kappaB, which was blocked by MG-132, and also reduced CD40L and IL-12 expression. CONCLUSIONS: This is the first report to demonstrate that bioactive surgical sutures can generate a prolonged T(H)1 immune response and expand flu-specific T cells. Bioactive sutures, which are primarily a T-cell stimulant, also stimulated other cells to secrete IL-12 and prolonged the immune response. Sutures may provide a novel in situ stimulating strategy for enhancing the immune system of cancer patients.

Adult↗

PubMed1981

Differential effects of somatostatin on the release of bioactive and immunoactive rat growth hormone in vitro.

The effects of linear (L) or cyclic (C) somatostatin (SRIF) on the release of growth hormone (GH) as measured both by bioassay and by radioimmunoassay (RIA) were studied in a series of incubation experiments with rat adenohypophyses. Although both forms of SRIF inhibited release of RIA-GH in all experiments, only C-SRIF appeared to inhibit release of bioactive GH. L-SRIF even enhanced release of bioassayable GH in one study. Addition of C-SRIF to medium containing secreted GH strongly inhibited the tibial growth response of the bioassay animals, while it had no effect on the measurement of GH by the RIA. Thus, the apparent inhibitory effect of C-SRIF on release of bioactive GH may be an artifact. The conclusion was supported by experiments in which the SRIF was not removed from the medium prior to assay by dialysis or by neutralization with anti-SRIF antiserum. After these manipulations, the inhibition by C-SRIF of the secretion of bioactive GH was eliminated, whereas the inhibitory effect on RIA-GH release was not affected. These results support other evidence for the differential release of rat GH with varying immuno-and bioactivities by different stimuli.

Animals↗

PubMed1996

An investigation into the lability of the bioactivity of human growth hormone using the ESTA bioassay.

We compared the bioactivity attributable to human growth hormone (hGH) in serum samples, determined at the time of their collection, with that after storage for 2-18 months at -20 degrees C. The samples were obtained from volunteers and patients who underwent provocative tests of hGH secretion, and the bioactivity was determined in the ESTA bioassay, which is based upon the use of Nb2 cells. We report that, in some subjects, the bioactivity of samples collected at the response peaks deteriorated on storage for as little as 2 months. The decrease in hGH bioactivity was systematic in that it consistently declined so as to approach the values initially determined by an immunoassay (Hybritech IRMA). This differential lability was a characteristic of the peak samples, and was not observed for either samples collected before and after the peaks of hGH secretion or for purified preparations of hGH which were subjected to a range of freeze/thaw and storage regimens. We suggest that this unusual lability is indicative of transient shifts in the spectrum of the variants of hGH which are present in the circulation following stimulation by provocative agents. This study emphasises the need to minimise the risk of introducing storage artefacts in investigations into the responses of hGH to provocation.

Adult↗

PubMed2005

Aldehyde dehydrogenase 2 plays a role in the bioactivation of nitroglycerin in humans.

OBJECTIVE: Nitrates are used widely in clinical practice. However, the mechanism underlying the bioactivation of nitrates to release NO remains unclear. Recent animal data suggest that mitochondrial aldehyde dehydrogenase (ALDH2) plays a central role in nitrate bioactivation, but its role in humans is not known. We investigated the role of ALDH2 in the vascular effects of nitroglycerin (NTG) in humans in vivo. METHODS AND RESULTS: Forearm blood flow (FBF) responses to intra-arterial infusions of NTG, sodium nitroprusside (SNP), and verapamil were measured in 12 healthy volunteers before and after ALDH2 inhibition by disulfiram. All drugs caused a dose-dependent vasodilatation. However, only the response to NTG was significantly reduced after disulfiram therapy (33% reduction in area under the curve [AUC]; P=0.002). Separately, 11 subjects of East Asian origin, with the loss-of-function glu504lys mutation in the ALDH2 gene, received intra-arterial NTG, SNP, and verapamil. Only the FBF response to NTG was lower in the volunteers with the glu504lys mutation compared with East Asian and non-Asian wild-type control subjects (40% reduction in AUC; P=0.02). CONCLUSIONS: The findings suggest that ALDH2 is involved in the bioactivation of NTG in humans in vivo but accounts for less than half of the total bioactivation. This may be of clinical importance in patients with mutations in the ALDH2 gene and in those taking drugs that inhibit ALDH2.

Adult↗

PubMed1999

All ApoB-containing lipoproteins induce monocyte chemotaxis and adhesion when minimally modified. Modulation of lipoprotein bioactivity by platelet-activating factor acetylhydrolase.

Mildly oxidized LDL has many proinflammatory properties, including the stimulation of monocyte chemotaxis and adhesion, that are important in the development of atherosclerosis. Although ApoB-containing lipoproteins other than LDL may enter the artery wall and undergo oxidation, very little is known regarding their proinflammatory potential. LDL, IDL, VLDL, postprandial remnant particles, and chylomicrons were mildly oxidized by fibroblasts overexpressing 15-lipoxygenase (15-LO) and tested for their ability to stimulate monocyte chemotaxis and adhesion to endothelial cells. When conditioned on 15-LO cells, LDL, IDL, but not VLDL increased monocyte chemotaxis and adhesion approximately 4-fold. Chylomicrons and postprandial remnant particles were also bioactive. Although chylomicrons had a high 18:1/18:2 ratio, similar to that of VLDL, and should presumably be less susceptible to oxidation, they contained (in contrast to VLDL) essentially no platelet-activating factor acetylhydrolase (PAF-AH) activity. Because PAF-AH activity of lipoproteins may be reduced in vivo by oxidation or glycation, LDL, IDL, and VLDL were treated in vitro to reduce PAF-AH activity and then conditioned on 15-lipoxygenase cells. All 3 PAF-AH-depleted lipoproteins, including VLDL, exhibited increased stimulation of monocyte chemotaxis and adhesion. In a similar manner, lipoproteins from Japanese subjects with a deficiency of plasma PAF-AH activity were also markedly more bioactive, and stimulated monocyte adhesion nearly 2-fold compared with lipoproteins from Japanese control subjects with normal plasma PAF-AH. For each lipoprotein, bioactivity resided in the lipid fraction and monocyte adhesion could be blocked by PAF-receptor antagonists. These data suggest that the susceptibility of plasma lipoproteins to develop proinflammatory activity is in part related to their 18:1/18:2 ratio and PAF-AH activity, and that bioactive phospholipids similar to PAF are generated during oxidation of each lipoprotein. Moreover, LDL, IDL, postprandial remnant particles, and chylomicrons and PAF-AH-depleted VLDL all give rise to proinflammatory lipids when mildly oxidized.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1987

Intrinsic bioactivity of thyrotropin in human serum is inversely correlated with thyroid hormone concentrations. Application of a new bioassay using the FRTL-5 rat thyroid cell strain.

We have developed a new bioassay for thyrotropin (TSH) in human serum to evaluate bioactivity in normal individuals and patients with different degrees of primary hypothyroidism. Unpurified TSH in serum showed no stimulation of cyclic AMP production in cultured FRTL-5 rat thyroid cells, but after immunopurification showed potent stimulatory activity. Immunoaffinity purification permitted up to 400-fold concentration of serum TSH, allowing bioactivity measurements even in certain normal sera. The limit of detection in the FRTL-5 bioassay was 10 microU of human TSH per 0.5 ml incubate, and half-maximal responses for standard human TSH was 102 +/- 26 (+/- SE) microU/0.5 ml. Immunoaffinity-purified serum TSH varied in bioactivity-to-immunoactivity (B/I) ratios from less than 0.25 to 1.21 among four euthyroid subjects and eight primary hypothyroid patients. An inverse correlation was found between B/I ratios of immunopurified basal TSH and the serum-free T4 (r = -0.7237, P less than 0.01), T4 (r = -0.6650, P less than 0.05), and T3 (r = -0.6382, P less than 0.05). B/I ratios of immunopurified TSH from three hypothyroid patients before and after acute stimulation by thyrotropin-releasing hormone showed no significant change, despite major changes in serum TSH. In summary, the present study shows an inverse relationship between the metabolic status of an individual and the intrinsic bioactivity of TSH.

Animals↗

PubMed1990

Serum prolactin bioactivity and immunoactivity in hyperprolactinaemic states.

Serum prolactin concentrations were measured using a sensitive bioassay (Nb2 assay) and by radioimmunoassay in 11 patients with prolactin-secreting pituitary tumours (median serum immunoactive prolactin 5150 mU/L), and in 58 normal control subjects (median prolactin 190 mU/L). The mean ratio of serum prolactin bioactivity to immunoactivity was significantly lower in patients with prolactinomas than in normoprolactinaemic controls. Ten lactating women in the early post-partum period (median prolactin 3800 mU/L), studied as a model of physiological hyperprolactinaemia, also had reduced bioactivity to immunoactivity ratios. Overall, there was a significant negative correlation between bioactivity:immunoactivity ratio and serum immunoactive prolactin. The change in relative bioactivity of prolactin in serum samples from patients with prolactinomas and in women with physiological hyperprolactinaemia may reflect changes in the molecular heterogeneity of the hormone. Such changes may affect activity in both bioassays and radioimmunoassays.

Adult↗