P. van den Broek

Determination of four metabolites of the plasticizer di (2-ethylhexyl) phthalate in human urine samples

SummaryA method for biological monitoring of exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) is described. In this method the four main metabolites of DEHP [i.e., mono (2-ethylhexyl) phthalate (MEHP), mono (5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)-phthalate] are determined in urine samples. The procedure includes enzymatic hydrolysis, ether extraction, and derivatization with triethyloxonium tetrafluoroborate. Analysis is performed by gas chromatography electron impact mass spectrometry. The detection limit for all four metabolites is less than 25 μg/l urine. The coefficient of variation based on duplicate determinations of urine samples of workers occupationally exposed to DEHP was 16% for MEHP (mean concentration 0.157 mg/l) and 6% -9% for the other three metabolites (mean concentrations 0.130-0.175 mg/1). The method described here was used to study DEHP metabolism in man. Most persons excrete mono(2-ethyl-5-oxohexyl)-phthalate and mono (2-ethyl-5-hydroxyhexyl)phthalate as a (glucuronide) conjugate. Mono (5-carboxy-2-ethyl-pentyl)phthalate is mainly excreted in free form, while for MEHP a large interindividual variation in conjugation status was observed. Of the four metabolites quantified, 52% are products of a ((ω-l)-hydroxylation reaction of MEHP [i.e., mono (2-ethyl-5-oxohexyl)phthalate and mono (2-ethyl-5-hydroxyhexyl)phthalate], 22% is the product of a ω-hydroxylation reaction of MEHP [i.e., mono (5-carboxy-2-ethylpentyl)phthalate], and 26% is not oxidized further (i.e., MEHP). A good correlation is obtained when the amount of MEHP ω-hydroxylation products is compared with the amount of MEHP (ω-1)hydroxylation products in urine samples. When the internal dose of DEHP has to be established we recommend that the levels of all four metabolites of DEHP be studied in urine samples.

Metabolites of the plasticizer di(2-ethylhexyl)phthalate in urine samples of workers in polyvinylchloride processing industries

SummaryLittle is known about occupational exposure to the plasticizer di(2-ethylhexyl)phthalate (CAS number 117-81-7), a compound widely used in polyvinylchloride (PVC) plastics. We have studied the uptake of DEHP in workers by determining the concentrations of four metabolites of DEHP in urine samples, i.e., mono(2-ethylhexyl)phthalate (MEHP), mono (5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)phthalate. In addition DEHP concentrations in the air were determined by personal air sampling. Nine workers in a PVC boot factory exposed to a maximum of 1.2 mg/m3 DEHP showed an increase in the urinary concentrations of all four metabolites over the workshift. These results were obtained on both the first and the last day of the workweek. With the exception of MEHP, the increases in the concentrations of the metabolites during a workday were statistically significant. Six workers from a PVC cable factory exposed to a maximum of 1.2 mg/m3 DEHP showed a one-to fourfold increase in the concentrations of the four metabolites over the workshift, but these increases were not statistically significant. These results indicate that measurement of DEHP metabolites in urine samples may be of use for monitoring the occupational exposure to DEHP.

The effect of short-term ventilation tubes versus watchful waiting on hearing in young children with persistent otitis media with effusion: a randomized trial.

Objective To study the effect of short-term ventilation tubes in children aged 1 to 2 yr with screening-detected, bilateral otitis media with effusion (OME) persisting for 4 to 6 mo, as compared with watchful waiting. Design Multi-center randomized controlled trial (N = 187) with two treatment arms: short-term ventilation tubes versus watchful waiting. Young children underwent auditory screening; those with persistent (4 to 6 mo) bilateral OME were recruited. Results The mean duration of effusion over 1-yr follow-up was 142 days (36%) in the ventilation tube (VT) group versus 277 days (70%) in the watchful waiting (WW) group. After 6 mo of follow-up, the pure-tone average in the VT group was 5.6 dB A better than that in the WW group. After 12 mo, most of the advantage in the VT group had disappeared. After the insertion of ventilation tubes, the children with poorer hearing levels at randomization improved more than the children with better hearing levels. The largest difference in hearing levels was found between the children in the VT group whose ventilation tubes remained in situ and the children in the WW group. In the VT children with recurrence of OME, the hearing levels again increased, but remained slightly lower than those in the infants with persistent OME in the WW group. Conclusions Ventilation tubes have a beneficial effect on hearing in the short run (6 mo); this effect, however, largely disappears in the long run (12 mo). This is probably due to partial recurrent OME in the VT group and to partial spontaneous recovery in the WW group.

The risk of vestibular function loss after intracochlear implantation

Sixty patients were selected for cochlear implantation and 50 of them received an intracochlear implant (Nucleus). Vestibular function was evaluated before and after surgery using a caloric test and a velocity step test. Sixteen patients had normal or residual vestibular function before surgery, 11 bilateral and 5 unilateral; in 3 of the latter patients, the ear with vestibular areflexia was elected for implantation, which reduced the number of patients at risk for vestibular dysfunction to 13. Vestibular function was preserved in all of these patients except for 4; the risk of vestibular function loss can therefore be rated at about 31%.

Vestibular Function in Cochlear Implant Patients

Thirty-five patients receiving a cochlear implant were evaluated using vestibular function tests. Twenty-five patients received an intracochlear implant (Nucleus). Three out of 6 patients with normo- or hyporeflexia before implantation showed postoperative vestibular damage. In one case this was iatrogenic. Together with available data from the literature the risk of losing preoperative vestibular function is estimated to be around 60%. Improvement of implantation techniques can probably reduce this risk considerably.

The relation between electric auditory brain stem and cognitive responses and speech perception in cochlear implant users.

Electrically evoked brainstem responses (EABR) and event-related cortical potentials were recorded in seven postlingually deaf adults who were experienced users of a Nucleus multichannel cochlear implant. The patients were divided into two subgroups: good performers and moderate performers. Poor EABR were found in two of the moderate performers. The latencies and amplitudes of the cortical N1 P2 complex in the good performers were within the same range as those of subjects with normal hearing, but were deviant in the group of moderate performers. This may indicate disturbed cochleotopical organization of the auditory cortex in the latter group. P300 measurements in the good performers showed normal latencies, whereas in the moderate performers they were prolonged. The results suggest that the outcomes of electrophysiological measurements to assess the integrity of a patients auditory neural system on a brainstem and a cortical level, are related to the patients performance with the cochlear implant.

Effects of the peroxisome proliferator mono(2-ethylhexyl)phthalate in primary hepatocyte cultures derived from rat, guinea pig, rabbit and monkey: Relationship between interspecies differences in biotransformation and peroxisome proliferating potencies

Primary hepatocyte cultures derived from rat, rabbit, guinea pig and monkey have been treated in vitro with metabolites of di(2-ethylhexyl)phthalate, i.e. mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (metabolite V) and mono(2-ethyl-5-oxohexyl)phthalate (metabolite VI). In rat hepatocyte cultures MEHP and metabolite VI were equally potent in inducing peroxisome proliferation, while metabolite V was much less potent. In rat hepatocytes a 50% increase in both peroxisomal palmitoyl-CoA oxidase activity and microsomal lauric acid omega-hydroxylation activity was found after treatment with 5-15 microM MEHP. In guinea pig, rabbit and monkey hepatocyte cultures, a 50% increase in peroxisomal palmitoyl-CoA oxidase activity was found after treatment with 408-485 microM MEHP. No induction of lauric acid omega-hydroxylation activity was found. These results indicate that peroxisome proliferation can be induced by MEHP in rabbit, guinea pig and monkey hepatocytes, but that these species are at least 30-fold less sensitive to peroxisome proliferation induction than rats. The proposed mechanistic inter-relationship between induction of lauric acid omega-hydroxylation activity and peroxisome proliferation is found in rat hepatocytes, but not in hepatocytes of the other three species. Treatment of guinea pig hepatocyte cultures with MEHP resulted in an increase in triglyceride concentrations in the hepatocytes. In rat and rabbit hepatocyte cultures, triglyceride concentrations were much less altered by MEHP. In monkey hepatocytes a decrease in hepatic triglyceride concentration was found after treatment with MEHP. These effects are in agreement with in vivo effects observed before. After treatment of primary hepatocyte cultures with MEHP, high concentrations of omega- and (omega-1)-hydroxylated metabolites of MEHP were found in media from rat, rabbit and guinea pig cultures. The formation of these metabolites did not decline in time. During treatment the metabolite profile in media from rat hepatocyte cultures moved towards omega-hydroxy metabolites of MEHP. In media from monkey hepatocyte cultures the lowest concentrations of hydroxylated metabolites were determined. No major species differences were found in the potency to form oxidized MEHP metabolites, and thus no unique metabolite differences were found, which could explain the species differences in sensitivity for peroxisome proliferation.

Effect of di(2-ethylhexyl)phthalate on enzyme activity levels in liver and serum of rats

In order to identify non-invasive, biochemical indicators of di(2-ethylhexyl)phthalate (DEHP) exposure, we have compared the effects in blood serum with biochemical effects in liver in rats fed a diet containing 0, 0.25, 0.75 and 2% DEHP for 2 weeks. After 3 days of treatment serum arylesterase activity levels and serum triglycerides were decreased to 60% and 20% of control values, respectively. After a 2-week treatment with DEHP the effects were generally stronger. Compared to a control group, serum arylesterase activity levels, serum triglycerides and serum cholesterol were decreased to 40%, 20% and 50%, respectively. Serum cholinesterase activity levels and serum albumin concentrations were increased by the DEHP treatment to 290% and 135% of control values, respectively. In the livers a hepatomegaly, an induction of cytochrome P-450 IVA1 and induction of the activity of palmitoyl-CoA oxidase and carnitine acetyl-CoA transferase was found to be 180%, 1080%, 1300% and 1700% of control values, respectively. The liver is a more sensitive target for DEHP exposure compared to the biochemical effects in serum, but determination of the serum parameters can be used to determine early biological effects of exposure to DEHP.

Surgical treatment of infantile subglottic hemangioma

Three cases of infantile subglottic hemangioma are described. Generally conservative management is favored by most authors. These present lesions were removed surgically by midline cricotracheotomy. Follow-up showed that all children were well and without complaints. The authors believe that besides management with the CO2 laser and short-term steroid therapy, operative treatment of infantile subglottic hemangioma is a valuable alternative which should receive more attention. All children were decannulated a few days after surgery.

Endothelial CD276 (B7-H3) expression is increased in human malignancies and distinguishes between normal and tumour-derived circulating endothelial cells.

Background:Mature circulating endothelial cells (CEC) are surrogate markers of endothelial damage. CEC measured in patients with advanced cancer are thought not only to derive from damaged normal vasculature (n-CEC), but also from damaged (t-CEC). Therefore, assays that allow the discrimination between these two putative types of CEC are thought to improve the specificity of the enumeration of CEC in cancer.Methods:Identification of tumour-associated endothelial markers (TEM) by comparing antigen expression on normal vs t-CEC and assess the presence of t-CEC in peripheral blood of cancer patients by incorporating TEM in our novel flow cytometry-based CEC detection assay.Results:No difference in antigen expression between normal and malignant endothelial cells (ECs) was found for CD54, CD109, CD137, CD141, CD144 and CXCR7. In contrast, overexpression for CD105, CD146, CD276 and CD309 was observed in tumour ECs compared with normal ECs. CD276 was most differentially expressed and chosen as a marker for further investigation. CD276-expressing CEC were significantly higher in 15 patients with advanced colorectal cancer (median 9 (range 1–293 cell per 4 ml); P<0.005), in 83 patients with a glioblastoma multiforme (median 10 (range 0–804); P<0.0001) and in 14 patients with advanced breast cancer (median 14 (range 0–390) P<0.05) as compared with 24 healthy individuals (median 3 (range 0–11)). Of all patients with malignancies, 58% had CD276+ CEC counts above the ULN (8 cell per 4 ml).Conclusions:The present study shows that CD276 can be used to discriminate ECs from malignant tissue from ECs from normal tissue. In addition, CD276+ CEC do occur in higher frequencies in patients with advanced cancer.