Determination of homovanillic acid and vanillylmandelic acid in neuroblastoma screening by stable isotope dilution GC-MS.

A method for the quantitative determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA), two metabolites of catecholamines, is presented. The assay is based on gas chromatography/electron impact mass spectrometry. The preparation of 13C-labeled VMA from [13C6]vanillin is described. Together with purchased deuterated HVA the 13C-labeled VMA is used as an internal standard in stable isotope dilution GC/MS. The method involves elution from soaked filter-papers, determination of creatinine content, extraction of HVA and VMA from eluted and urinary samples and derivatization to the di-and tri(trimethylsilyl) derivatives, respectively. The detection limits were found to be 4.0 pg for HVA and 0.8 pg for VMA. The method was applied to the routine determination of urinary HVA and VMA in a range from 5 to 100 ng HVA and VMA per microgram creatinine. The lower limits of pathological concentrations are set at 35 ng micrograms-1 creatinine for HVA and to 20 ng micrograms-1 creatinine for VMA, which are in close correlation with the values from other methods, but with the main advantage of reducing the amount of questionable or elevated results from 6.7% (high-performance liquid chromatography (HPLC) alone) to 0.9% (HPLC and GC/MS).

Phenotypic heterogeneity of osteoblast-like MC3T3-E1 cells: changes of bradykinin-induced prostaglandin E2 production during osteoblast maturation.

We have examined clonal murine calvarial MC3T3-E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin-induced production of PGE2). It was found that the sublines investigated showed large variation of the above-mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above-mentioned agents. The most striking difference was observed with bradykinin-induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of 3H-bradykinin to its receptor and also correlated well with the bradykinin-induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast-like cell line MC3T3-E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

Method for the determination of vitamin K1(20) in human plasma by stable isotope dilution/gas chromatography/mass spectrometry.

A method for the quantitative determination of vitamin K1(20) (VK), an essential cofactor in the carboxylation of clotting factors, is presented. The assay is based on gas chromatography/electron impact mass/spectrometry. The preparation of deuterium-labelled vitamin K1(20) for use as an internal standard is described. The method involves extraction of VK from human plasma and its derivatization to the heptafluorobutyryl ester after reduction of one carbonyl group with zinc. The detection limit was found to be 1.0 pg and the limit of quantitation 2.0 pg ml-1 plasma. This permits the measurement of vitamin K1(20) even in small quantities of plasma, which is highly desirable in investigations dealing with clotting abnormalities in neonates and infants.

Inhibition of prostanoid formation in intact cells by 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a blocker of Ca(2+)-ATPases.

1 The blocker of endoplasmic reticulum Ca(2+)-ATPase, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) was shown to inhibit formation of prostaglandin E2 and prostacyclin in the osteoblast-like cell lines, MC3T3-E1 and ROS 17/2.8, respectively, in a dose-dependent manner with an IC50 of 0.5-1 microM. Inhibition was observed with various stimuli (arachidonic acid, bradykinin, melittin and calcium ionophore, A23187). 2 This effect was also observed in human platelets, where BHQ dose-dependently blocked thromboxane biosynthesis and formation of 12-hydroxy-heptadecatrienoic acid after stimulation with arachidonic acid, but not formation of 12-hydroxy-eicosatetraenoic acid. 3 Inhibition of prostaglandin E2 formation in MC3T3-E1 cells was not observed with thapsigargin after stimulation with arachidonic acid, A23187 or melittin, whereas bradykinin-induced prostaglandin E2 biosynthesis was blocked. 4 Taken together, the results suggest a direct inhibitory action of BHQ on the cyclo-oxygenase in these three cell systems.

[5 years neuroblastoma screening in Austria: rate of participation, results and a comparison with other screening areas]. / Fünf Jahre Neuroblastom-Screening in Osterreich: Beteiligungsrate, Ergebnisse und ein Vergleich mit anderen Screeningregionen.

In late 1990 a screening program for the early detection of neuroblastoma in infants was introduced in Austria. The program is performed on a voluntary basis in collaboration with general pediatricians and practitioners. Filter strips for urine collection are distributed to parents of infants aged seven to nine months on the occasion of a routine check up. The samples are sent to the laboratory by parents and analysed for vanillylmandelic acid (VMA) and homovanillic acid (HVA). Between January 1991 and December 1995 125,201 infants were screened. The compliance rate was 26.8% for Austria, but great differences were seen for different regions (65% in Carinthia, 10% in Vorarlberg). 30 children were admitted to hospital for investigation of repeatedly elevated urine catecholamines. A neuroblastoma was identified in 16 cases. In 12 of these cases at least one unfavorable prognostic factor was present (stage > or = 3, elevated LDH, unfavorable histology, N-myc amplification, di- or tetraploidy). Neuroblastoma screening of infants aged more than six months seems to detect predominantly those tumors which are unlikely to regress spontaneously. The observation of one false negative case, however, demonstrates that neuroblastomas which become clinically manifest at a later date may remain undetected by early screening. Possible advantages of shifting screening to a later age and repeated screening are discussed.

Neuroblastoma screening: labeling of HVA and VMA for stable isotope dilution gas chromatography-mass spectrometry.

Neuroblastoma is a curable tumor if detected early enough. Therefore, a screening test is performed in Austrian infants by analyzing urine samples collected and dried on filter strips. Screening is performed by measuring homovanillic acid (HVA) and vanillylmandelic acid (VMA) using the enzymatic immunoassay chemistry (EIA) method and reexamining the elevated values by high-performance liquid chromatography. Both methods, however, give a relatively high number of false-positive results. Therefore, the positives are reexamined by stable isotope dilution (SID) gas chromatography-mass spectrometry (GC-MS), which is the most sensitive and precise method in clinical chemistry. The labeled substance is added as an internal standard to the sample; it has nearly identical chemical and physical properties as the natural analogue, undergoes the entire preparation step, and is detected simultaneously with the natural analogue by the same detector, namely the mass spectrometry, given by a difference in molecular weight. All loss is compensated in this way by the final calculation. At present, HVA but not VMA can be purchased labeled with deuterium, so we started with our method using deuterated HVA as the internal standard for both HVA and VMA. Because the coefficient of variation is not sufficient for VMA, we try to label VMA via different labeling procedures; for example, 1) exchange of the aromatic H against D by incubation with DCI or in alkaline R-OD or LiOD or 2) exchange of phenolic 16OH against 18OH by reaction with H2(18)O in an acidic environment and at high temperatures. Carboxylic 16O can be exchanged against 18O.

On the inhibition of prostanoid formation by SK&F 96365, a blocker of receptor-operated calcium entry.

1. The proposed blocker of receptor-operated calcium channels, SK&F 96365 was shown to inhibit formation of prostaglandin E2 in two osteoblast-like cell lines, MC3T3-E1 and UMR-106 in a dose-dependent manner at an IC50 of 3-4 microM. Inhibition was observed with various stimuli (arachidonic acid, bradykinin and calcium ionophore A23187). 2. This effect was also observed in human platelets, where SK&F 96365 dose-dependently blocked thromboxane biosynthesis and formation of 12-hydroxy-heptadecatrienoic acid after stimulation with arachidonic acid (IC50 = 4 microM). 3. The compound had no effect on 12-hydroxy-eicosatetraenoic acid production by human platelets. Additionally, linoleic acid oxidation by soybean 15-lipoxidase was not impaired by SK&F 96365. The results thus provide evidence for cyclo-oxygenase inhibition by SK&F 96365 at concentrations used to block receptor-operated calcium influx.

Extracellular Ca2+ sensing by the osteoblast-like cell line, MC3T3-E1.

The present study was undertaken to clarify the role of extracellular calcium on osteoblast activation. It was found that bradykinin and thrombin induced synthesis of prostaglandin E2 was strongly dependent on the concentration of extracellular calcium in the osteoblast-like cell line, MC3T3-E1. Moreover, this effect was not related to Ca2+ influx, since it was even potentiated by Ni2+ and Co2+, which was not due to intracellular activity of Ni2+, as judged by studies with 63Ni2+. Ba2+, Mg2+ and Sr2+ had no effect. Cd2+ caused dose-dependent synthesis of prostaglandin E2, which was shown to correlate with its cytotoxic properties. The results thus strongly suggest the presence of a divalent cation sensor in osteoblast-like MC3T3-E1 cells.

Quantitative measurement of N-acetyl-L-aspartic acid in urine by gas chromatography with negative-ion chemical ionization mass spectrometry.

A highly specific and sensitive assay for N-acetyl-L-aspartic acid has been developed. The trideuterated compound was synthesized and used as an internal standard for gas chromatography with negative-ion chemical ionization mass spectrometry. Urine samples were acidified and extracted with ethyl acetate, and the compounds converted into their pentafluorobenzyl ester derivatives. Under these conditions, sub-picogram amounts of the pure derivatives could be detected. Thus, only microliter volumes of urine samples have to be processed to achieve reliable quantification of "basal" levels of N-acetyl-L-aspartic acid.

A patient with propionic acidemia managed with continuous insulin infusion and total parenteral nutrition.

An infant newly diagnosed with propionic acidemic coma was managed successfully with total parenteral nutrition (TPN) and continuous infusion of insulin. The urinary excretion of 3-hydroxypropionic acid was reduced to 3% of the admission value in 4 days, gradually decreasing to 1.5% in 16 days. The treatment did not prevent a prolonged episode of thrombocytopenia. The infant tolerated TPN well, except for continued hyper-lactic acidemia (2 to 4 times normal). Metabolic acidosis and mild hyperammonemia recurred only when the patient had sepsis secondary to Candida albicans and Staphylococcus aureus infection.

Eicosanoid synthesis in the isolated human renal pelvis, ureter and bladder.

Prostaglandin synthetase inhibitors are used for the treatment of ureteric colic. However, there is controversy regarding the mechanism of action of these drugs. In this study, differential prostaglandin synthesis in the human renal pelvis, ureter and bladder was measured using specific radioimmunoassays and gas chromatography/mass spectrometry. There was a significant quantitative predominance of the smooth muscle constrictor eicosanoids, PGF2 alpha and TXA2 over the dilatory PGE2 in tissue from all sites--renal pelvis, ureter and bladder. The results indicate that prostaglandins play a direct role in smooth muscle activity of the upper urinary tract and the inhibition of this activity with indomethacin indicates a further mode of its action in pain relief in ureteric colic.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase deficiency in Saudi Arabia.

Deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase has been studied in 11 Saudi infants. The diagnosis was established by the measurement of enzyme activity in lymphocytes, in fibroblasts and, in seven patients, by the gas chromatography/mass spectrometer pattern of excreted organic acids in the urine. In seven infants the disease caused a devastating acidotic attack within the first day of life, while in two the crisis occurred by the third day of life. In two infants from one family the disease appeared later in infancy. The clinical presentation of an acidotic attack is lethargy, hyperpnoea, tachypnoea and seizures, either at birth (two infants), following first feeding (in five infants), or following vomiting or refusal of food in later infancy. The acidotic attacks recurred later in life following minor illness or refusal to eat. The acidosis of this enzyme deficiency progresses rapidly, leading to cardiopulmonary arrest and death within hours of onset unless treated promptly. In four surviving infants diagnosed and treated early, development is normal. Magnetic resonance and computerized tomography brain scans in these infants, however, show white matter lesions and mild atrophy.

Quantitative determination of terbutaline and orciprenaline in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry.

A method for the determination of unconjugated terbutaline and orciprenaline in human plasma is described. The assay is based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry. An inexpensive and rapid method for preparation of stable isotope labelled analogues as well as their use in quantitative gas chromatography/mass spectrometry is shown. A highly efficient sample work-up procedure with product recoveries of more than 95% is presented. The method developed permits quantitative measurement of terbutaline and orciprenaline in human plasma down to 100 pg ml-1, using 1 ml of sample. Plasma levels of terbutaline after oral administration of 5 mg of terbutaline sulphate were estimated.

Bone-resorbing activity of thyroid hormones is related to prostaglandin production in cultured neonatal mouse calvaria.

The bone-resorbing activity of thyroid hormones was evaluated in neonatal mouse calvaria maintained in organ culture for 96 h. Thyroxine (T4) between 10(-8) and 10(-5) mol/liter and triiodothyronine (T3) between 10(-8) and 10(-7) mol/liter caused a dose-dependent release of calcium from cultured bone. The thyroid hormone effect was delayed in onset for at least 24 h, and after 96 h of culture amounted to 50-90% of the bone-resorbing activity of 10(-8) mol/liter parathyroid hormone (PTH). The bone-resorbing action of T4 as well as of T3 was completely blocked by 100 U/ml interferon-gamma (IF-gamma) or 20 mU/ml salmon calcitonin (CT). "Escape" from CT inhibition, which is a well-known phenomenon in the action of PTH, was not observed with thyroid hormone-mediated bone resorption. Thyroid hormone treatment of cultured calvaria resulted in a gradual increase between 48 and 96 h of medium concentrations of prostaglandin (PG) E2 and particularly of 6-keto-PGF1 alpha, the inactive metabolite of prostacyclin (PGI2). The release of PGF2 alpha in general was not significantly affected. Although the effect of thyroid hormones on PG release from cultured calvaria was completely abolished by 5 x 10(-7) mol/liter indomethacin, in some experiments indomethacin reduced thyroid hormone-mediated bone resorption by only 50%. This indicates that thyroid hormone action on bone is also mediated by a PG-independent mechanism.

Effect of auranofin on resorption, prostaglandin synthesis and ultrastructure of bone cells in cultured mouse calvaria.

Auranofin (AF) in concentrations between 3 x 10(-7) and 3 x 10(-6) mol/l stimulated bone resorption in cultured neonatal mouse calvariae significantly with 1 x 10(-6) mol/l being most potent. Complete inhibition by 5 x 10(-7) mol/l indomethacin and increased medium concentrations of prostaglandin (PG) E2 and 6-keto-PGF1 alpha after 72 h indicate a PG mediated mechanism. Morphology revealed active osteoclasts. Cytotoxic effects were observed with 3 x 10(-6) and 1 x 10(-5) mol/l AF with osteocytes and osteoblasts being considerably more sensitive than osteoclasts. The latter concentrations inhibited bone resorption stimulated by parathyroid hormone (PTH) 1,25-dihydroxyvitamin D3, PGE2, thrombin and interleukin 1. The stimulatory effect of AF on PG production and subsequent bone resorption could limit its therapeutic usefulness.

Isotope dilution gas chromatography-mass spectrometry for the study of eicosanoid metabolism in human blood platelets.

Stable isotope dilution gas chromatography-mass spectrometry provides one of the most important techniques for the quantitative measurement of eicosanoids. This technique was applied to the quantitation of hydroxyeicosatetraenoic acids, hydroxyheptadecatrienoic acid, thromboxane B2 and prostaglandin F2 alpha formed during platelet aggregation after stimulation of gel-filtered platelets with thrombin (0.25 U/ml) or collagen (2 micrograms/ml). Similar amounts of hydroxyheptadecatrienoic acid and thromboxane B2 were found after platelet activation. The ratio of formation of 12-hydroxyeicosatetraenoic acid to thromboxane B2 varied from donor to donor. Only small amounts of prostaglandin F2 alpha (up to 200 pg per 2.0.10(8) platelets) and basic values of 15-hydroxyeicosatetraenoic acid (up to 100 pg per 2.0.10(8) platelets) were measured using gas chromatography with negative ion chemical ionization mass spectrometry. In addition, different stable isotope dilutions were prepared and are discussed in detail.

Femtomole analysis of diclofenac in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry using (18O2) diclofenac as internal standard.

A stable isotope dilution gas chromatographic/negative ion chemical ionization mass spectrometric assay for diclofenac in human plasma is described. The preparation of (18O2)diclofenac and its use as an internal standard for quantitative gas chromatography/mass spectrometry is shown. A sample processing and derivatization sequence with product recovery of 84.7% was found. The method presented permits quantitative measurement of diclofenac in human plasma at the lower femtomole level. Plasma levels of diclofenac after administration of diclofenac gel were estimated.