Fat and fish intake and asthma in Japanese women: baseline data from the Osaka Maternal and Child Health Study.

OBJECTIVE: It remains controversial whether the intake of n-3 polyunsaturated fatty acids and fish is preventive against asthma. This cross-sectional study investigated the relationship between fat and fish intake and the prevalence of asthma using baseline data from a prospective study. DESIGN: The subjects were 1002 pregnant Japanese females. A diet history questionnaire was used to assess dietary habits. Current asthma and asthma after age 18 were defined as present if subjects had been treated with medications at some time in the previous 12 months and after reaching the age of 18, respectively. RESULTS: Fish consumption was independently associated with a decreased prevalence of asthma after age 18 and current asthma. A significant inverse relationship was observed between the ratio of n-3 to n-6 polyunsaturated fatty acid intake and the prevalence of current asthma, but not asthma after age 18. Intake of total fat, saturated, monounsaturated, n-3 polyunsaturated and n-6 polyunsaturated fatty acids, cholesterol, meat, eggs or dairy products was not evidently related to either outcome for asthma. CONCLUSION: Our results suggest that fish consumption and the high ratio of n-3 to n-6 polyunsaturated fatty acid intake may be associated with a reduced prevalence of asthma in young female Japanese adults.

Magnesium intake is inversely associated with the prevalence of tooth loss in Japanese pregnant women: the Osaka Maternal and Child Health Study.

There have only been a few studies on the role of mineral intake in tooth loss. We investigated the association between mineral intake and the prevalence of tooth loss in Japan. We used the baseline data on 1002 pregnant women who were enrolled in the Osaka Maternal and Child Health Study between November 2001 and March 2003. Tooth loss was defined as the previous extraction of one or more teeth. Nutrient intake was assessed by a validated diet history questionnaire. Prevalence odds ratios and confidence intervals were estimated by applying a multiple logistic regression model. The adjusted odds ratio upon comparison of the highest quartile with the lowest quartile of magnesium intake was 0.64 (95% confidence interval, 0.42-0.99), showing a tendency for an inverse dose-response relationship (p for linear trend = 0.05). There were no associations between the level of consumption of calcium, phosphate, iron, zinc, or copper and tooth loss. The present findings suggest that intake of magnesium is related to reduced prevalence of tooth loss among young Japanese women.

Relationship between active and passive smoking and total serum IgE levels in Japanese women: baseline data from the Osaka Maternal and Child Health Study.

BACKGROUND: Many studies have shown that cigarette smoking is associated with elevated concentrations of total serum IgE. Few studies, however, have examined total IgE in relation to passive smoking exposure, especially in adults. This cross-sectional study investigated the association of active and passive smoking exposure with levels of total serum IgE in Japan. METHODS: Study subjects were 981 pregnant women in Osaka. Total IgE levels were measured using UniCAP 1000 and were defined as elevated if they exceeded 170 ml/UI. Age, gestation, parity, family history of asthma, atopic eczema and allergic rhinitis, indoor domestic pets, family income, education and the mite allergen level in house dust were selected as potential confounding factors. RESULTS: Current smoking of at least 15 cigarettes a day and 8.0 or more pack-years of smoking were independently related to an increased prevalence of elevated total serum IgE (adjusted odds ratios 3.40 and 2.51, 95% confidence intervals 2.12-5.47 and 1.55-4.06, respectively), and both cigarette smoking status and pack-years of smoking were significantly positively associated with total serum IgE levels, especially in subjects with a positive familial allergic history. There was no measurable association of exposure to environmental tobacco smoke (ETS) at home or at work with total serum IgE concentrations among those who had never smoked. CONCLUSIONS: Our results corroborate a positive relationship between active smoking and total serum IgE levels; however, this study failed to substantiate a positive association of ETS exposure with total IgE. Investigations with more precise and detailed exposure measurements are warranted.

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450.

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.

Hemadsorption expressed by cloned H genes from subacute sclerosing panencephalitis (SSPE) viruses and their possible progenitor measles viruses isolated in Osaka, Japan.

Most subacute sclerosing panencephalitis (SSPE) viruses, including our Osaka-1, -2, and -3 strains isolated in Osaka, have shown negative hemadsorption (HAD) by African green monkey red blood cells. This property has been thought to be characteristic of SSPE virus as compared to the positive reaction of the standard Edmonston strain of measles virus (MV). However, this assumption has become quite obscure because MV mutates frequently at the genetic level during its multiplication and also because recent field strains isolated by lymphoblastoid cell lines have shown negative HAD. To investigate the above issue, the nucleotide sequences of the hemagglutinin (H) genes from SSPE virus Osaka-1, -2, or -3 strains were compared to those of various MV field strains isolated in Osaka by Vero cells. The H gene sequences of three SSPE strains were relatively conserved without such biased hypermutation as had been observed in the matrix (M) gene of three SSPE strains. However, this analysis of the H gene sequence of the SSPE viruses enabled us to deduce possible progenitor MVs, which are in agreement with the deduction from the M gene analysis we reported previously. The HAD of Vero cells transfected with the cloned H cDNAs from the SSPE strains and their progenitors suggested that negative HAD of the SSPE viruses has been maintained as one of original properties of the progenitor MVs rather than having been acquired as an altered one during long-term persistent infection in the brains of patients with SSPE.

Unique heme environment at the putative distal region of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis (peroxygenase P450(SPalpha).

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is a hydrogen peroxide-dependent cytochrome P450 (P450) enzyme (P450(SPalpha)). In this study, heme-ligand exchange reactions of P450(SPalpha) were investigated using the optical spectroscopic method and compared with those of various P450s. Alkylamines (C >/= 5) induced changes in the spectrum of ferric P450(SPalpha) to one typical of a nitrogenous ligand-bound low-spin form of ferric P450, although their affinities were lower than those for other P450s, and a substrate, laurate, did not interfere with the binding in contrast with in the cases of other P450s. Other compounds having a nitrogen donor atom to the heme iron of P450, including pyridine or 1-methylimidazole, induced no change in the spectrum of P450(SPalpha) in either the ferric or ferrous state. Practically no spectral change was observed on the addition of alkyl isocyanides to ferric P450s. On the other hand, cyanide induced a change in the spectrum of ferric P450(SPalpha) to one characteristic of cyanide-bound form of ferric P450. The affinity of cyanide increased when the substrate was added, in contrast with in the cases of other P450s. Ferrous P450(SPalpha) combined with CO and alkyl isocyanides, and the affinity for CO was of the same order of magnitude as in the cases of other P450s. These findings suggest a unique heme environment of P450(SPalpha), in which most compounds usually acting as external ligands of ferric P450s are prevented from gaining access to the heme iron of P450(SPalpha). The unique properties of the hydroxylase reaction catalyzed by P450(SPalpha), where an oxygen atom of hydrogen peroxide but not of molecular oxygen is utilized and incorporated into a fatty acid at its alpha position, is possibly related with such a specific heme environment of this P450. A possible mechanism for the peroxygenase reaction of P450(SPalpha) is proposed.

A lung model describing uptake of organic solvents and roles of mucosal blood flow and metabolism in the bronchioles.

A simple lung model (mucosal blood flow and metabolism model, MBM model) was developed to describe the uptake of organic solvents and investigate the role of mucosal blood flow and metabolism. The model separates the lung into four compartments, the peripheral bronchial tract (gas phase), the mucus layer lining the wall surface of the tract, the alveolar space (gas phase), and the alveolar blood. Solvent molecules are absorbed in the mucus layer during inhalation and released during exhalation. The deposited solvent diffuses radially into the mucosal tissue of the respiratory tract and transfers to the mucosal blood flow. To describe this behavior, a hypothetical mucosal blood flow throughout the mucus layer was used. The solvent in the mucosal tissue may be also metabolized, and a hypothetical metabolism in the mucus layer was used. The rate of the hypothetical mucosal blood flow was determined to be 5.2 ml/min based on the best fitting of previously obtained data for seven polar organic solvents. The MBM model predicts that as the blood-air partition coefficient (lambda(B)) increases from 0.1 to 20, the relative end-exhalation (E(end)) will decrease from 0.89 to 0.07, and as lambda(B) increases to 500, E(end) will increase to 0.33. After lambda(B) = 500, E(end) is predicted to decrease again, and at lambda(B) = 10000, E(end) is 0.09. The model also predicts that as lambda(B) increases from 0.1 to 10, the relative uptake (U) increases from 0.08 to 0.61, and as lambda(B) increases to 150, U decreases to 0.50. After lambda(B) = 150, U increases again, and at lambda(B) = 10,000, U is 0.8. The predictions show good agreement with values observed in human experimental studies. The MBM model predicts that uptake by the mucosal blood (U(Al)) would be equal to uptake by the alveolar blood (U(Mu)) at lambda(B) of 1000 and U(Al) is more than 90% of total uptake at lambda(B) > 10,000. The model also shows that U is significantly increased by the mucosal metabolism at lambda(B) between 50 and 5000. Especially, U in the case of CL(Mu) = 100 ml/min is higher by 0.3 than that in the nonmucosal metabolism.

Fatty acid-specific, regiospecific, and stereospecific hydroxylation by cytochrome P450 (CYP152B1) from Sphingomonas paucimobilis: substrate structure required for alpha-hydroxylation.

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the alpha-carbon of fatty acids in the presence of H2O2. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of the alpha-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon chain lengths of more than 10. The Km value for pentadecanoic acid (C15) was the smallest among the saturated fatty acids tested (C10-C18) and that for myristic acid (C14) showed similar enzyme kinetics to those seen for C15. As shorter or longer carbon chain lengths were used, Km values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of magnitude (10(3) min(-1)), but the turnover number for undecanoic acid (C11) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and omega-hydroxypalmitic acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C14 or C15. The metabolite of arachidonic acid was only alpha-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were not utilized as substrates. Analysis of the optical configurations of the alpha-hydroxylated products demonstrated that the products were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of omega-carbon and carboxyl carbon as well as carbon chain length of fatty acids are important for substrate-enzyme interaction.

Cell surface expression of immature H glycoprotein in measles virus-infected cells.

Two forms of hemagglutinin (H) protein, one with an apparent molecular mass of 78 kDa (78K H protein) and the other with that of 74 kDa (74K H protein), are present in cells infected with measles virus (MV). We previously observed that only the mature 78K H protein, a completely glycosylated form of the 74K H protein, was expressed on the cell surface of the infected cells. In the present study, we detected transient expression of the 74K H protein on the cell surface of infected cells by pulse-chase studies, although the level of this expression was much lower than that of the 78K H protein. On the cell surface the 74K H protein was present as dimers and sensitive to endo-beta-N-acetylglucosaminidase H digestion. Treatment with brefeldin A, which blocks the transport of membrane and secretory proteins from the endoplasmic reticulum to the Golgi apparatus, inhibited the cell surface expression of the 78K H protein, but not that of the 74K H protein. These data suggest that a part of the MV 74K H proteins could be transported directly to the cell surface - probably via an alternative pathway - without processing to the complex form in the Golgi apparatus.

Characterization of the ybdT gene product of Bacillus subtilis: novel fatty acid beta-hydroxylating cytochrome P450.

We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis. In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product. The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450. In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce beta-hydroxymyristic acid and alpha-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry. The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture. The amount of beta-hydroxyl product was slightly higher than that of alpha-hydroxyl product at all times during the reaction. However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used. HPLC analysis with a chiral column showed that the beta-hydroxyl product was nearly enantiomerically pure R-form. These results suggest that this P450 enzyme is involved in a novel biosynthesis of beta-hydroxy fatty acid.

An epidemic of acute haemorrhagic conjunctivitis caused by enterovirus 70 in Okinawa, Japan, in 1994.

BACKGROUND: Although enterovirus 70 (EV70) has been identified as the major aetiological agent of acute haemorrhagic conjunctivities (ACH),no EV70 strain has been isolated by cell culture method since 1988. Therefore, recent clinical and epidemiological characteristics of AHC caused by EV70 have not been clarified. METHODS: Clinical and serological studies were carried out on patients during the AHC epidemic in Okinawa, Japan, in 1994 in which 7509 cases were reported by national epidemiological surveillance. EV70 was confirmed as the causative agent by reverse-transcription polymerase chain reaction. RESULTS: The 11-15 years age group contained the highest number of cases (62% of the total). Conjunctival hyperaemia was present in all patients, and subconjunctival haemorrhage, superficial punctate keratitis and preauricular lymphadenopathy were present in 24.0%, 11.7% and 9.3% of AHC cases, respectively. No neurological complication was observed in this epidemic. Out of 31 paired serum samples, 10 pairs showed a fourfold rise in antibody level to EV70. None of the paired serum samples showed a fourfold rise in antibody level to Coxsackie A24 variant virus. CONCLUSION: These findings demonstrate that the clinical features of AHC observed in this study were milder than those reported previously, in contrast to the high transmission rate during an epidemic. Changes in clinical features of AHC, such as a low incidence of subconjunctival haemorrhage and disappearance of neurological complications, might be due to biological transformation of EV70. It should be noted that EV70 is still an important aetiological agent of explosive epidemics of AHC.

Uptake of 10 polar organic solvents during short-term respiration.

Respiratory uptake was investigated for 10 polar organic solvents with high blood/air partition coefficients (lambda(blood/air)): ethyl acetate (lambda(blood/air), 77), methyl iso-butyl ketone (90), methyl acetate (90), methyl propyl ketone (150), acetone (245), iso-pentyl alcohol (381), iso-propyl alcohol (848), methyl alcohol (2590), ethylene glycol monobutyl ether (EGBE, 7970), and propylene glycol monomethyl ether (PGME, 12380). Test-air concentrations (Cinh) were 25 to 200 ppm. Four healthy male volunteers inhaled the test air for 10 min at rest and then room air for 5 min. The percentage of solvent in the end-exhaled air and in the mixed-exhaled air increased after the start of the test-air respiration, and reached a quasi-steady-state level within a few min. The speeds of these increases at the start of the test-air respiration became lower as lambda(blood/air) increased. The mean uptakes (U) for the last five min of the test air respiration were 67.3, 52.9, 60.4, 53.0, 52.6, 63.0, 60.3, 60.8, 79.7, and 81.3%, respectively, for ethyl acetate, methyl iso-butyl ketone, methyl acetate, methyl propyl ketone, acetone, iso-pentyl alcohol, iso-propyl alcohol, methyl alcohol, EGBE and PGME. Thus, U values of the alcohols were higher than those of the ketones and lower than the glycol ethers. The overall view, except for esters, showed that U increased with lambda(water/air) increases. This tendency can be explained by a hypothesis that solvent absorbed in the mucus layer of the respiratory tract is removed by the bronchial blood circulation. U values of ethyl acetate and methyl acetate were higher than those of methyl iso-butyl ketone and methyl propyl ketone, though the lambda(blood/air) values of these esters were nearly equal to those of the ketones. For the respiration of the esters, their metabolites, ethyl alcohol and methyl alcohol, were detected in the exhaled air. The exhalation percentage of the metabolites increased after the start of test-air respiration and reached a quasi-steady-state level of 2 and 3%, respectively, by the 5th min. These data suggest that removal of the solvent via metabolism in the wall tissue of the respiratory tract plays an important role for the esters.

Within-shift variability of short-term exposure to organic solvent in indoor workplaces.

This study examined the degree of within-shift variability of short-term exposure concentrations for workers exposed to organic solvents in indoor workplaces. For this purpose, 117 exposure data sets of 15-minute time-weighted average (15-min TWA) and those of 60-min TWA were collected from 53 workers employed in the offset printing, gravure printing, screen printing, machine control board production, fiber-reinforced plastic production, hard metal production, electrical parts production, and chemical synthesizing industries. Data analysis showed that the tenth, fiftieth, and ninetieth percentiles of the geometric standard deviations of 15-min TWA values [GSD(15m)] were 1.4, 2.3, and 4.5, respectively; and those of GSD(60m) were 1.2, 1.7, and 3.4, respectively. Based on an assumption of lognormal exposure distribution, the maximum values of 15-min TWA (the 98.4th percentile) were estimated to be 4.3, 36, and 650 times as high as the minimum one (the 1.6th percentile) for the low, middle, and high exposure variabilities, respectively; and to be 2.0, 4.3, and 8.2 times the 8-hour TWA value, respectively. Consequently, when the 8-hour TWA exceeds 0.23 times (1/4.3) the short-term exposure limit value, the high short-term exposure condition should be evaluated. The maximum values of 60-min TWA (the 93.8th percentile) were estimated to be 1.8, 5.1 and 43 times as high as the minimum one, respectively; and to be 1.3, 2.0, and 3.1 times the 8-hour TWA value, respectively. The relationship between production factors and within-shift exposure variability was also examined. The intermittent solvent use group had significantly higher median values of GSD(15m) and GSD(60m) than the continuous group. The mobile pollutant source group had a significantly higher median value of GSD(60m) than the stationary group.

[Load on the low back of teachers in kindergartens].

In order to evaluate the load on the low back of teachers in kindergartens, basic activity and working posture were analyzed for four teachers by means of video recording. The trunk inclination angle (TIA) was also measured continuously during full workshifts for 12 kindergarten teachers by means of an inclination monitor. The kindergarten teachers spent 67% of the workshift on activities in contact with children, "indoor group childcare", "indoor free playing", "outdoor childcare", "preparation and clearing away" and "help and care", and did not take a recess during the workshift. They spent 36% of the workshift in three working postures with the load on the low back, "standing bent forward", "squatting" and "kneeling". Cumulative time at a TIA of 20 degrees or more represented 43% of the workshift. The frequency of trunk-lifting from severe bending forward (TIA > 45 degrees) was 95 times/hr on average. A comparison of the kindergarten teachers and nursery teachers in 4-5 year age classes showed that the time distributions of basic activities were generally similar to each other. Although the time distributions of working postures were also similar, time spent "standing bent forward", "squatting" and "kneeling" was longer in the kindergarten teachers than in the nursery teachers. Cumulative time at a TIA of 45 degrees or more was significantly longer in the kindergarten teachers. Although the frequency of trunklifting was not significantly different, the kindergarten teachers tended to lift their trunk more frequently. The present study found that the load on the low back was considerably great in the kindergarten teachers.

[A proposed method for evaluating short-term exposure condition].

High short-term exposure to toxic chemicals can occur during a workday, even if the daily average exposure is lower than the permissible exposure limit, because the exposure concentration varies from minute to minute. To protect workers from acute health effects due to high short-term exposure, the Japan Society for Occupational Health recommends that the maximum value for 15-min time-weighted average (15-min TWA) exposure during a workday should not exceed 1.5 times the occupational exposure limit for 8-hr TWA, and the American Conference of Governmental Industrial Hygienists issues the threshold limit value-short-term exposure limit (TLV-STEL), that is a 15-min TWA exposure which should not be exceeded at any time during a workday. A workday (8 hr) consists of thirty two 15-min periods. If the thirty-two 15-min TWAs are measured, the short-term exposure situation can be appropriately evaluated by comparing the highest measured value with the standard value (e.g. TLV-STEL), but such continuous monitoring consumes a lot of cost and time. In this paper, we propose a method for evaluating short-term exposure by using three or more measured values. This evaluation method corresponds to two different types of selection of sampling periods. One is a random selection of three or more 15-min periods among the 32 periods. If this selection is adopted, a comparison between the 98.44 percentile of the within-day distribution of 15-min TWAs and the standard value can be made by using one-sided tolerance factors, KI, KII and KIII, and the exposure situation is classified into four exposure classes at 95% and 50% confidence levels. Another is a random selection among high exposure periods. If this selection is adopted, a comparison between the specific percentile of the distribution and the standard value can be made with modified one-sided tolerance factors, and the exposure class is determined similarly. This method can provide a precise evaluation of exposure, so that it is useful in the industrial hygiene field.

Further characterization of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Although fatty acid alpha-hydroxylase (FAAH) activity has been detected in various species, FAAH has not been sufficiently characterized. In this report, we describe the properties of FAAH highly purified from Sphingomonas paucimobilis. The FAAH was purified by about 5,200-fold. Blotting analysis with a specific antibody against the FAAH showed that its apparent molecular mass was approximately 43 kDa. FAAH showed alpha-hydroxylation activity in the presence of H2O2, but little if any activity with cumene hydroperoxide, t-butyl hydroperoxide, or t-butyl peroxybenzonate. The Km value for H2O2 was 72 microM. Highly purified FAAH oxidized various non-esterified saturated and unsaturated fatty acids including myristic acid, but not myristoyl-CoA. Potassium cyanide and sodium azide inhibited the FAAH activity in a concentration-dependent manner. Other respiratory chain inhibitors such as rotenone and antimycin A did not inhibit the activity. Among cytochrome P450 inhibitors, SKF-525A markedly inhibited the activity at the concentration of 2 mM, but CO did not. Imidazole, an inhibitor of plant alpha-oxidation, showed no inhibitory effect at 1 mM.

Effects of variation in exposure to airborne acetone and difference in work load on acetone concentrations in blood, urine, and exhaled air.

Using a physiologically based pharmacokinetic (PBPK) model, the effects of variation of exposure concentration of acetone on three biological indicators--acetone concentrations in blood, urine, and exhaled air--were investigated. The effect of the difference in work load was also examined. It was confirmed that the model could be used to estimate acetone concentrations during fluctuating exposure by comparing simulated acetone concentrations with the corresponding values observed in field surveys. By inputting the exposure situations into the PBPK model, the variabilities of the biological indicators were simulated. The variation of acetone exposure was expressed by seven 1-hour time-weighted averages (CEXPs). The arithmetic means of the CEXPS were 200 and 750 ppm. The geometric standard deviations (GSDs) were 1.5, 2.0, and 3.0, representing low, moderate, and high variations, respectively. Work loads were set at 15 and 50 W. Consequently, there were 12 exposure situations. The acetone concentrations in venous blood (CB) and exhaled alveolar air (CA) at 1 minute after the end of the work shift were selected as biological indicators of exposure because they were predicted to decrease rapidly at the end of exposure and become relatively stable after 1 minute. The acetone concentration in urine excreted during the last 2 hours of the work shift (CU) was also used as a biological indicator. Simulation was repeated 100 times with randomly permuting CEXPs for each situation. The mean values of CB, CU, and CA showed almost no variation regardless of the difference in the GSD of CEXPs. The coefficients of variation increased with the GSD of CEXPs but were less than 0.2. Consequently, these variables were acceptable as biological indicators of daily average exposure for the same work load. However, the difference in work load greatly changed the mean values of CB, CU, and CA, thus making it difficult to use these variables as indicators of daily average exposure for different work loads.

Phytanic acid alpha-hydroxylation by bacterial cytochrome P450.

Fatty acid alpha-hydroxylase, a cytochrome P450 enzyme, from Sphingomonas paucimobilis, utilizes various straight-chain fatty acids as substrates. We investigated whether a recombinant fatty acid alpha-hydroxylase is able to metabolize phytanic acid, a methyl-branched fatty acid. When phytanic acid was incubated with the recombinant enzyme in the presence of H2O2, a reaction product was detected by gas chromatography, whereas a reaction product was not detected in the absence of H2O2. When a heat-inactivated enzyme was used, a reaction product was not detected with any concentration of H2O2. Analysis of the methylated product by gas chromatography-mass spectrometry revealed a fragmentation pattern of 2-hydroxyphytanic acid methyl ester. By single-ion monitoring, the mass ion and the characteristic fragmentation ions of 2-hydroxyphytanic acid methyl ester were detected at the retention time corresponding to the time of the product observed on the gas chromatogram. The Km value for phytanic acid was approximately 50 microM, which was similar to that for myristic acid, although the calculated Vmax for phytanic acid was about 15-fold lower than that for myristic acid. These results indicate that a bacterial cytochrome P450 is able to oxidize phytanic acid to form 2-hydroxyphytanic acid.

Molecular cloning and expression of fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Fatty acid alpha-hydroxylase (FAAH) catalyzes the initial reaction in alpha-oxidation of fatty acid to produce 2-hydroxy fatty acid. FAAH activity has been detected in a wide range of organisms from prokaryotes to eukaryotes. Here, we describe cloning of the FAAH gene from Sphingomonas paucimobilis, a sphingolipid- and 2-hydroxymyristic acid-rich bacterium. The isolated gene encoded 415 amino acids. A homology search revealed that amino acid sequences highly conserved in cytochrome P450 (P450) were present in FAAH. Although the heme-binding cysteine was recognizable at position 361, the consensus in the heme-binding region was modified by an insertion. Overall, FAAH has no significant identity to the known P450s. CO difference spectrum of recombinant FAAH showed the characteristic one of P450, except this peak was at 445 nm. These results suggest bacterial FAAH is a novel member of the P450 superfamily.

Quantitative determination of urinary metabolites of o-dichlorobenzene using a gas chromatograph.

o-Dichlorobenzene (o-DCB) is used as an organic solvent, as a chemical intermediate, and as a heat transfer medium. In humans, o-DCB is metabolized to 2,3- and 3,4-dichlorophenols, and 3,4- and 4,5-dichlorocatechols, and these metabolites are eliminated via the kidneys. In this study, we tried to determine the concentrations of urinary 2,3- and 3,4-dichlorophenols using a gas chromatograph (GC). When control urine specimens were spiked at concentrations of 10, 20 and 40 mg/l, the mean recovery rates of 2,3- and 3,4-dichlorophenols were 98.3 to 101.9% (CV = 4.0 to 4.8%) and 100.6 to 105.4% (CV = 2.5 to 7.0%), respectively. Next, urine samples collected from ten male workers exposed to o-DCB were analyzed. The concentrations of urinary 2,3- and 3,4-dichlorophenols determined by the GC method closely agreed with those by the HPLC method, which we had developed in a previous study, and these metabolite concentrations were linearly correlated to the 8-h TWA values of o-DCB in the range of 0.1 to 2.3 ppm. Consequently, the GC method can be used for biological monitoring of o-DCB, though it is necessary that the linear relation is confirmed in a higher range of exposure.