Determination of methyl 5-hydroxy-2-benzimidazole carbamate in urine by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic (HPLC) assay for methyl 5-hydroxy-2-benzimidazole carbamate (5-HBC) in urine was developed in order to assess the exposure of workers to the pesticide carbendazim. 5-HBC is measured in urine after hydrolysis, sample clean-up through a strong cation-exchange (SCX) column and extraction with ethyl acetate. HPLC with electrochemical detection provides selective and sensitive determination of 5-HBC with a detection limit of 5 micrograms/l. A C18 reversed-phase column was used with 0.06 M ammonium acetate solution (pH 8)-methanol (73:27) as mobile phase. The method was validated with respect to hydrolysis of urine samples, analytical recovery of spiked 5-HBC, stability of 5-HBC conjugates, limit of detection, background and precision. The overall analytical recovery from urine was better than 60%. 5-HBC, excreted in urine as a conjugate, was stable for at least one year when stored at -20 degrees C. A background of ca. 5 micrograms/l was detected in urine from some non-occupationally exposed persons. Between-day coefficients of variation as calculated from the results of the stability test were 7, 4 and 4% for concentrations of 61, 244 and 295 micrograms/l 5-HBC, respectively (n = 16).

Determination of 2-isopropoxyphenol in urine by capillary gas chromatography and mass-selective detection.

An analytical method for the assessment of the exposure of workers to the pesticide propoxur through biological monitoring has been developed. This study was part of a survey of occupational exposure to pesticides used in greenhouses for the growing of ornamental plants. In order to assess the actual absorbed amount of propoxur in the body, an analytical method for its metabolite 2-isopropoxyphenol in urine was required. This led to the development of a gas chromatographic-mass spectrometric assay involving hydrolysis and solvent extraction. A mass-selective detector, operated in single-ion mode, provides a selective and sensitive quantification of 2-isopropoxyphenol with a detection limit of 6 micrograms/l. The method has been validated with respect to the hydrolysis of urine samples, analytical recovery of 2-isopropoxyphenol, stability of its conjugates, limit of detection, background and precision. The analytical recovery from spiked urine was over 95%. 2-Isopropoxyphenol was excreted in urine as a conjugate and was stable for at least seven months when stored at -20 degrees C. It was not detected in urine from non-exposed persons. Between-day coefficients of variation were 20, 10, 7 and 4% for concentrations of 15, 29, 150 and 213 micrograms/l, respectively. Measured as 2-isopropoxyphenol, ca. 80% of an orally administered dose of propoxur was excreted in urine within 10 h.

Gas chromatographic determination of the pesticide dodemorph for assessment of occupational exposure.

As part of a health hazard survey of occupational exposure to pesticides in greenhouse growing of ornamentals, analytical methods are developed and validated for measurement of exposure of workers to the fungicide dodemorph. A gas chromatographic method is developed using on-column injection and nitrogen-phosphorus detection for quantification. Methods for the determination of (potential) dermal exposure by the analysis of foliar dislodgeable residues and cotton gloves are validated with respect to background, analytical recovery, stability, limit of detection, and between-day coefficients of variation. Analytical recovery from 'foliar dislodgeable residue solutions' and cotton gloves is more than 95%. Dodemorph in 'foliar dislodgeable residue solutions' and on cotton gloves is stable for at least five and six months, respectively, when stored in the refrigerator. Between-day coefficients of variation are 6% for both matrices. The limit of detection is 3 micrograms per leaf sample and 150 micrograms per pair of gloves. Institute of Occupational Medicine (IOM) samplers, designed for the collection of a defined inspirable fraction of aerosols, are tested for sampling air-borne dodemorph. IOM samplers equipped with glass-fiber or cellulose filters appear unsuitable for reliable sampling of the fungicide because of breakthrough or breakdown during sampling.

Assessment of dermal exposure of greenhouse workers to the pesticide bupirimate.

An HPLC method was developed for estimation of dermal exposure of greenhouse workers to the pesticide bupirimate. Chromatography was performed on a cyano-modified silica column with methanol-water (6:4 by volume) containing 5 g/L ammonium sulfate as eluent. UV detection at 310 nm was used for quantitation. Dermal exposure was assessed by letting the workers wear cotton gloves and by measuring foliar dislodgable residues in the greenhouses as potential exposure. The analytical procedure was validated for measurement of bupirimate on cotton gloves and in solutions used for the estimation of foliar dislodgable residues. Gloves were extracted with methanol. Recovery of bupirimate from fortified gloves was complete. Methanol extracts with one volume of water added and solutions containing dislodgable residues were injected directly onto the HPLC system. The limit of detection was 30 micrograms/L. Between-day coefficients of variation were 7 and 4% at concentrations of 0.6 and 28 mg/L, respectively.

An automated method for the determination of acetyl and pseudo cholinesterase in hemolyzed whole blood.

The aim of the present study was to develop a method which allows determination of pseudo (PsChE) and acetyl cholinesterase (AChE) activities in single hemolyzed blood samples of workers exposed to cholinesterase-inhibiting compounds, avoiding the time-consuming and laborious separation of plasma and erythrocytes. Two methods based on Ellman's colorimetric determination of cholinesterase activity were compared, and three different substrates were tested. The best results were obtained with the substrates butyrylthiocholine and acetyl(beta-methyl)thiocholine, both showing a substrate specificity of more than 97% with respect to PsChE and AChE, respectively. The method showed sensitivity to detect low levels of inhibition of AChE and PsChE in blood. The between-day precision was less than 4% for both cholinesterase activities. It was demonstrated with this method that hemolyzed blood can be stored at -20 degrees C at least 18 months without loss of cholinesterase activity. The method has been used for 18 months in a monitoring program for laboratory employees working with various cholinesterase-inhibiting compounds. The average co-efficients of intraindividual variation amounted to 6.8% (range 2.2-9.6%; 90 percentile, 8%) and 6.6% (range 2.9-9.9%; 90 percentile, 7.9%) for PsChE and AChE, respectively. In a group of non-exposed workers the average intraindividual variations were 4.0% (range 1.5-7.7%; 90 percentile, 7.6%) and 3.6% (range 0.6-6.6%; 90 percentile, 5.3%), respectively. Using the value of 4.0%, it appears possible to detect an individual decrease in cholinesterase activity of more than 8% below a baseline based on three determinations. The method can thus be used to detect relatively low levels of cholinesterase inhibition.

High-performance liquid chromatographic method for the determination of occupational exposure to the pesticide abamectin.

As part of a survey of occupational exposure to pesticides in greenhouses for growing ornamentals, analytical methods were developed and validated for the measurement of exposure of workers to the pesticide abamectin. Abamectin consists of a mixture of avermectin-B1a and avermectin-B1b, which are members of a class of fermentation products of the soil microorganism Streptomyces Avermitilis. Because of the high molecular weight of the avermectins (greater than 800 daltons), high-performance liquid chromatography (HPLC) was the analytical method of choice. Previously described HPLC methods that used fluorescence detection were adapted and validated for the determination of dermal exposure by the analysis of cotton gloves and foliar dislodgeable residue. IOM samplers (developed at the Institute of Occupational Medicine, Edinburgh, U.K.) for collecting the inspirable fraction of dust or aerosols were tested for the determination of airborne abamectin concentrations in greenhouses. An analytical procedure considerably simpler than published methods appeared suitable for the determination of abamectin residues on cotton gloves and on greenhouse foliage. Analytical recovery from cotton gloves, solutions of foliar dislodgeable residues, and air-sampling filters was essentially complete. However, air concentrations of abamectin could not be reliably measured by using the IOM sampling device because of breakdown during sampling. Between-day coefficients of variation for solutions of dislodgeable residue and cotton glove extracts were between 3% and 6% for abamectin concentrations between 5 and 140 micrograms/L.

Determination of the pesticide chlorothalonil by HPLC and UV detection for occupational exposure assessment in greenhouse carnation culture.

An HPLC method was developed for application in the measurement of occupational exposure to the pesticide chlorothalonil. In addition, sampling methods were validated for the determination of exposure to chlorothalonil in the greenhouse culturing of carnations. Procedures for sampling of the inspirable fraction of aerosols, for the determination of hand contamination by hand rinse and the use of cotton gloves, and for the determination of dislodgable chlorothalonil residues on carnation leaves were validated. Normal phase HPLC with hexane-dioxane as the eluent and UV detection at either 254 or 325 nm appeared suitable for the determination of chlorothalonil in the described matrices. The limit of detection, after concentration on SepPak C18 cartridges was approximately 0.5 micrograms/L. Linear calibration curves were obtained in concentration ranges from 0.5 microgram/L to 100 mg/L. In general, no interferences were noticed in the analysis of the matrices. However, cotton gloves for determination of hand contamination had to be washed before use because they contained interfering material, and in the case of air sampling, glass fiber filters for air sampling appeared to degrade chlorothalonil very rapidly. Therefore, cellulose filters were used for collection of the inspirable fraction of aerosols containing chlorothalonil.

The effect of vitamin D supplementation on vitamin D status and parathyroid function in elderly subjects.

Vitamin D deficiency is common in the elderly and may lead to secondary hyperparathyroidism, cortical bone loss, and hip fractures. The effect of vitamin D supplementation for 1 yr was studied in 72 people living in a nursing home and 70 people living in an aged people's home. The subjects were randomized into 3 groups: control, and 400 or 800 IU vitamin D3/day. The initial vitamin D status of each subject was classified as deficient or borderline [serum 25-hydroxyvitamin D (25OHD) less than 30 nmol/L] in 79% and adequate (serum 25OHD greater than or equal to 30 nmol/L) in 21%. Serum 25OHD concentrations increased about 3-fold in both groups receiving vitamin D supplementation. Serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations increased slightly but significantly, and the increase was inversely related to the initial serum 25OHD concentration. Serum intact PTH-(1-84) concentrations decreased about 15% during supplementation in both nursing home and aged people's home residents, whereas serum osteocalcin significantly decreased in the nursing home residents only. We conclude that a vitamin D3 supplement of 400 IU/day adequately improves vitamin D status in elderly people and increases 1,25-(OH)2D concentrations in those with vitamin D deficiency. Supplementation decreases parathyroid function and may depress bone turnover to some degree.

Improvements in the simultaneous determination of calcidiol and calcitriol in human serum or plasma.

Technical improvements have been applied to reduce the time required for the determination of calcidiol and calcitriol in serum or plasma. The modifications include the use of Sep-Pak C18 cartridges for the extraction of calciol metabolites from serum instead of a classical liquid/liquid extraction, a considerable shortening of the HPLC purification time compared with our previously described method, and the application of HPLC with UV detection at 254 nm of calcidiol as an alternative to the usual competitive protein binding methods. In none of the 199 samples where calcidiol was determined by HPLC did we observe a detectable peak of ercalcidiol. Quantitation of calcidiol by HPLC and competitive protein binding was compared in 5 series of assays. The correlation between the two methods was 0.99. The average slope of the linear regression line when the HPLC values were plotted versus the competitive protein binding values was 1.14. The average intercept was 0.19 nmol/l. The mean within-run coefficient of variation for calcidiol in these series was 5% for the competitive protein binding method, and 4% for HPLC method. Between-run coefficients of variation were 6% and 12% for the competitive protein binding and for the HPLC method, respectively. Within-run and between-run coefficients of variation for calcitriol were 6% and 15%, respectively.

Determinants of vitamin D status in patients with hip fracture and in elderly control subjects.

The factors that influence vitamin D status were investigated in 125 patients with hip fracture and in 74 elderly control subjects. The serum concentrations of 25-hydroxyvitamin D [25(OH)D] varied with sunshine score and were paralleled by serum 1,25-dihydroxyvitamin D [1,25(OH)2D]. The control subjects showed a higher sunshine score and higher serum 24(OH)D levels than the patients with hip fracture. Dietary vitamin D intake was similar in both groups (mean 115 IU/d). A positive correlation between vitamin D intake and serum 25(OH)D was observed in the patients with low sunshine exposure. It appeared from this relation that dietary vitamin D intake should be approximately 300 IU/d to maintain an adequate serum (25(OH)D concentration. Vitamin D status was very poor in patients who were institutionalized before hip fracture. Multiple regression analysis on serum 25(OH)D confirmed the primary role of sunshine exposure as determinant of vitamin D status. The principal determinants of serum 1,25(OH)2D were serum 25(OH)D, serum creatinine, and serum phosphate.

Effect of dietary calcium, phosphate and vitamin D deprivation on the pharmacokinetics of 1,25-dihydroxyvitamin D3 in the rat.

The in vivo regulation of circulating 1,25(OH)2D3 concentrations by vitamin D status and by dietary calcium and phosphate deficiency was studied. Adult rats were cannulated in the jugular vein and the clearance of physiological doses of 1,25(OH)2D3 monitored. In vitamin D-replete rats we investigated the effects of dietary calcium and phosphate deficiency on the elimination half life of 1,25(OH)2D3 The results showed no effect of dietary phosphate deficiency on the elimination half life of 1,25(OH)2D3. Dietary calcium deficiency resulted in a small increase of the 1,25(OH)2D3 elimination half life (P = 0.04) (normal diet: 16.3 +/- 1.8 hrs, n = 6; -Ca diet: 18.6 +/- 1.1 hrs, n = 5; -P diet: 16.0 +/- 1.4 hrs, n = 6; mean +/- SD). The experiments with the vitamin D deficient rats showed a marked increase in the elimination half life of 1,25(OH)2D3 (36.4 +/- 6.8 hrs, n = 7), when compared to the rats on the normal diet (P = 0.001). From the experiments in the vitamin D replete rats one can infer that regulation of circulating 1,25(OH)2D3 concentrations by dietary calcium or phosphate takes place at the production site and not by changes in elimination rate. However, vitamin D status appears to regulate circulating 1,25(OH)2D3 concentrations also through an effect on the elimination rate.

A simplified cannulation procedure for pharmacokinetic experiments in rats.

A convenient, simplified method for cannulating the jugular vein of rats is described. The technique has been developed for use in pharmacokinetic experiments. The construction of the cannula has been simplified compared with previously described methods. In addition, a small and simple outlet device for the cannula is described which allows the connection of tubing for sampling at short time intervals or for short-term infusions (several hours). In order to reduce stress on the animal, the tubing can be disconnected during sampling at long time intervals, allowing free movement of the animal with only a very small external outlet device.

Increased plasma calcitonin levels in young spontaneously hypertensive rats: role in disturbed phosphate homeostasis.

In young spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto controls (WKY) several parameters of phosphate and calcium homeostasis were determined. At 6 and 8 weeks, blood analysis revealed a significant hypophosphatemia (p less than 0.001) in SHR and twice as high plasma calcitonin levels in SHR than in WKY controls. At 8 weeks, 1,25-dihydroxycholecalciferol concentration was 20% higher in SHR (p less than 0.02) while 25-hydroxycholecalciferol was unaltered (p greater than 0.51). In addition total immunoreactive PTH, iPTH, was slightly increased (p less than 0.07) but intact PTH (1-84) (p greater than 0.90) was not significantly different from age matched WKY controls. Also at 8 weeks, a slightly reduced serum ionized Ca2+ concentration (p less than 0.001) with no change in total serum calcium was found in SHR (p greater than 0.39). Balance studies at 6 and 8 weeks of age revealed no significantly different balances for phosphate (F = 2.5, p greater than 0.10) and for calcium (F = 2.6, p greater than 0.09), although a tendency for slightly more positive balances existed in SHR when compared to WKY. However, SHR excreted significantly less phosphate in the urine than WKY control (F = 0.2, p less than 0.0009). Bone analysis was performed on femora of SHR and WKY of 6 weeks of age. Femora were significantly shorter in SHR (20.54 +/- 0.35 vs. 21.50 +/- 0.05 mm in WKY), whereas bone dry weight (127 +/- 6 vs. 107 +/- 2 mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of effect of 24,25-dihydroxyvitamin D3 in primary hyperparathyroidism.

Nineteen patients with primary hyperparathyroidism were treated with 25 micrograms 24,25-dihydroxyvitamin D3 or placebo daily for 3 months according to double-blind cross-over protocol. Serum immunoreactive PTH, total and ionized calcium, urinary calcium excretion, tubular reabsorption of phosphate/glomerular filtrate, and urinary hydroxyproline excretion did not change significantly. Serum 24,25-dihydroxyvitamin D3 levels increased significantly from 1.4 +/- 2.2 (SD) nmol/liter to 38 +/- 11 nmol/liter during the treatment period. Serum 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 levels did not change. We conclude that pharmacological doses of 24,25-dihydroxyvitamin D3 have no suppressive effect on parathyroid function in primary hyperparathyroidism.

Vitamin D status in urinary calcium stone formation.

Serum vitamin D metabolites were measured in 160 normocalcemic urinary calcium stone formers and in 217 control subjects. No difference in concentrations of 25-hydroxy-vitamin D (25[OH]D), 24,25-dihydroxyvitamin D (24,25[OH]2D), and 1,25-dihydroxyvitamin D (1,25[OH]2D) was found between stone formers and control subjects. Values for 25(OH)D and 24,25(OH)2D were higher in hypercalciuric stone formers than in normocalciuric stone formers independent of seasonal fluctuation. No difference in concentration of serum 1,25(OH)2D was found between hypercalciuric and normocalciuric stone formers. No correlations were present between the serum concentrations of the measured vitamin D metabolites and of measures of calcium and phosphate metabolism. These findings suggest no major pathophysiologic role of the main vitamin D metabolites in urinary calcium stone formation.

The effect of trauma on serum concentrations of vitamin D metabolites in patients with hip fracture.

In a previous study we observed lower serum concentrations of 25(OH)D, 24,25(OH)2D, and 1,25(OH)2D in patients with hip fracture than in aged control subjects. In order to evaluate the effect of trauma on vitamin D metabolite levels, we measured serum concentrations of vitamin D binding protein (DBP) in 118 patients with hip fracture and 71 aged control subjects. Serum DBP was lower in the patients than in the controls (mean +/- SD 315 +/- 60 vs 371 +/- 44 mg/l, P less than 0.001). Serum DBP correlated positively with serum total protein, albumin, alpha 2-globulin, and the vitamin D metabolite levels in the patients. When correcting for differences in serum DBP, serum 25(OH)D and 24,25(OH)2D still were significantly lower in patients than in controls, whereas serum 1,25(OH)2D was not. The free 1,25(OH)2D index (10(5) x molar ratio 1,25(OH)2D/DBP) was lower in patients than in controls, but the level of significance was marginal. This difference was not significant when patients and controls with impairment of renal function were excluded. It is concluded that the difference in serum 25(OH)D and 24,25(OH)2D between patients and controls is largely preexistent. However, the lower serum 1,25(OH)2D in the patients is mainly caused by the trauma. The free 1,25(OH)2D concentrations are almost similar in the two groups when renal function is normal.

An international comparison of vitamin D metabolite measurements.

An international 19-laboratory survey was organized to compare assays for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D in plasma. Each participant received two ethanolic standard solutions of each metabolite and eight plasma samples. Each laboratory used its usual procedures. Mean interlaboratory coefficients of variation (CVs) for the eight plasma samples were 35%, 43%, and 52% for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D, respectively. Average CVs for the standard solutions were 27%, 23%, and 25%, respectively. Of the eight plasma samples, five had the same concentration for one of the metabolites. One sample was diluted to 0.6 times its original concentration and three samples were fortified with one or more of the metabolites under investigation. Fourteen of 18 laboratories (78%) could distinguish between the five unchanged samples and the modified ones with their 25-hydroxyvitamin D assay. Nine of 12 (75%) could distinguish the modified samples from the other samples with the 24,25-dihydroxyvitamin D assay. Only eight of 15 (53%) could do this their 1,25-dihydroxyvitamin D assay. Values from different laboratories evidently cannot be intercompared without making an actual comparison of the assay procedures. Furthermore, in case of clinical applications of these assays, each laboratory should establish its own reference values and should continually use an internal reference sample to assess the precision of the procedures.

Measurement of vitamin D metabolites in anephric subjects.

Circulating concentrations of 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D and 25-hydroxyvitamin D were measured in 21 anephric subjects. 13 subjects had no therapy with vitamin D, dihydrotachysterol or 1 alpha-hydroxyvitamin D3. In 7 subjects of this group 1,25-dihydroxyvitamin D was undetectable (less than 5 pmol/l). In the other 6 patients concentrations ranged from 10 to 43 pmol/l (reference value 111 +/- 33 pmol/l). All subjects taking high doses of vitamin D showed detectable 1,25-dihydroxyvitamin D concentrations in the same range. Dihydrotachysterol therapy caused spuriously high '1,25-dihydroxyvitamin D' values, probably by interference of a metabolite of dihydrotachysterol in our assay. In subjects on vitamin D or dihydrotachysterol therapy 25-hydroxyvitamin D concentrations were significantly elevated (314 +/- 146 nmol/l and 98 +/- 19 nmol/l, respectively; reference value 52 +/- 22 nmol/l). Concentrations of 24,25-dihydroxyvitamin D were only measured in subjects without vitamin D2 intake. In general very low but detectable concentrations were found. One subject on a high dose of vitamin D3 showed a 24,25-dihydroxyvitamin D3 concentration of 10.2 nmol/l (reference value 4.4 +/- 2.9 nmol/l). Our results therefore confirm earlier reports on extrarenal synthesis of 24,25-dihydroxyvitamin D and suggest that there may be extrarenal production of 1,25-dihydroxyvitamin D as well.

Seasonal variation in serum concentrations of parathyroid hormone in elderly people.

In 124 patients with femoral neck fracture and 74 aged control subjects a seasonal variation was observed in the serum concentrations of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and PTH. The serum PTH concentrations were maximal in winter, when the vitamin D metabolites were lowest, suggesting a secondary phenomenon.

Interlaboratory variation of vitamin D1) metabolite measurements.

Interlaboratory variation of the measurement of 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D by six laboratories in the Netherlands was studied. Three different serum samples and two different standard solutions of each metabolite were assayed. Substantial interlaboratory variation was found for the measurement of serum samples. The mean interlaboratory CV's for the 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D assays in the three sera were 48%, 38% and 20% respectively. The measurement of standard solutions of all metabolites showed relative little variation (mean CV 8%). The small number of samples allowed no evaluation of intralaboratory variation. The much higher CV's of the measurement of serum samples, when compared to standard solutions, may be attributed to differences in extraction and purification procedures which are probably responsible for the presence of varying amounts of interfering substances during the final quantification of metabolites.