Determination of 2,5-hexanedione,a metabolite of n-hexane,in urine: evaluation and application of three analytical methods

Three methods for the determination of 2,5-hexanedione (2,5-HD) in urine were compared in order to assess their applicability for toxicokinetic studies and biological monitoring of occupational exposure to n-hexane. Two of them were based on derivatization, followed by gas chromatography and electron-capture detection. of these two, one is a modification of the other, already published, method. The third one involves direct extraction of 2,5-HD followed by gas chromatography and flame-ionization detection. To determine 2,5-HD in urine of workers occupationally exposed to n-hexane, the most straightforward method, direct extraction of 2,5-HD from urine, has been proven to be the most suitable. However, in case of very low concentrations of 2,5-HD in urine, or analysis of small samples of blood, e.g. in kinetic studies, it is necessary to use a more sensitive procedure. The sensitivity of the methods based on the derivation of 2,5-HD followed by electron-capture detection, was, as expected, much higher in terms of analytical reliability. By using these methods, however, precautions are necessary to avoid a matrix effect.     

Different Administration Schedules of the Same Dose of 2,5-Hexanedione Influence the Development of Neuropathy and the Toxicokinetics

The same total dose (1.2 g/kg/week) of 2,5-hexanedione (2,5-HD) was administered subcutaneously at 100 mg/kg/12 hr, 200 mg/kg/24 hr, and 400 mg/kg/48 hr to three groups of Donryu rats. The peripheral neuropathy induced by 2,5-HD was confirmed by clinical observation every day, and neurophysiological measurements every 4 weeks. During the 15th week of this experiment, 2,5-HD concentrations in plasma 0.5 to 24 hours after injection were determined. It was found that the greater the dose of 2,5-HD per treatment injected, the earlier peripheral neuropathy developed. Toxicokinetic analysis showed that both the values of the area under the plasma concentration versus time curve and the half life of 2,5-HD were increased, but the excretion parameters (Ke) were decreased, in animals treated with 200 mg/kg/24 hr and 400 mg/kg/48 hr 2,5-HD.     

Urinary 2,5 hexanedione as a biomarker of n-hexane exposure

n-Hexane is a saturated aliphatic hydrocarbon widely used in industry. In most cases it is used as a mixture with hexane isomers and various others solvents in the form of commercial hexane. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (2,5-HD), which is eliminated through the urine and is implicated in the neurotoxic effect of this solvent. The main objective of this study was to evaluate urinary 2,5-HD as a biomarker of n-hexane exposure. The study was carried out in seven industrial units. Post-shift urine samples from 111 workers who handled commercial hexane were collected and analysed for 2,5-HD by capillary gas chromatography. Air sampling was performed in the breathing zones of the workers, and the air samples were analysed using validated methods. Monitoring individual exposures showed that n-hexane exposure varied from 5 to 70 p.p.m. (mean±SD = 15.24±2.98 p.p.m.). Significant correlation was observed between exposure to n-hexane and urinary 2,5-HD levels, with high correlation coefficients (ρ= 0.81, p = 0.000), suggesting that urinary 2,5-HD is a good biomarker of occupational exposure to n-hexane. Urinary 2,5-HD is recommended as a better tool than air monitoring in the assessment of health risk, namely the early detection of n-hexane neurotoxicity.     

Urinary 2,5 hexanedione as a biomarker of n-hexane exposure

n-Hexane is a saturated aliphatic hydrocarbon widely used in industry. In most cases it is used as a mixture with hexane isomers and various others solvents in the form of commercial hexane. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (2,5-HD), which is eliminated through the urine and is implicated in the neurotoxic effect of this solvent. The main objective of this study was to evaluate urinary 2,5-HD as a biomarker of n-hexane exposure. The study was carried out in seven industrial units. Post-shift urine samples from 111 workers who handled commercial hexane were collected and analysed for 2,5-HD by capillary gas chromatography. Air sampling was performed in the breathing zones of the workers, and the air samples were analysed using validated methods. Monitoring individual exposures showed that n-hexane exposure varied from 5 to 70 p.p.m. (mean ±SD = 15.24 ±2.98 p.p.m.). Significant correlation was observed between exposure to n-hexane and urinary 2,5-HD levels, with high correlation coefficients ( ρ= 0.81, p = 0.000), suggesting that urinary 2,5-HD is a good biomarker of occupational exposure to n-hexane. Urinary 2,5-HD is recommended as a better tool than air monitoring in the assessment of health risk, namely the early detection of n-hexane neurotoxicity.     

Biological monitoring of n-hexane exposure in shoe workers

To analyse working conditions and to provide information about the degree to which shoe workers are exposed to n-hexane, the urinary excretion of total 2,5-hexanedione (2,5-HD) was determined in 81 employees in 12 shoe factories. Twenty-five individuals who had experienced no exposure to solvents were used as controls. 2,5-HD was measured in spot urine samples collected from workers at the end of shift. In the urine of shoe workers, the 2,5-HD presented a mean value of 2.33 mg g^-1 creatinine, a median of 1.96 mg g^-1 creatinine. The mean 2,5-HD concentration in the urine samples from non-exposed subjects was 0.28 mg g^-1 creatinine, the median value was 0.18 mg g^-1 creatinine. The mean time-weighted average (TWA) concentration of n-hexane in 12 shoe workshops was 126.1 ppm, ranging from 23 to 215 ppm. We found a significant, but low, correlation (r = 0.40; p < 0.001) between TWA intensity of environmental exposure to n-hexane and the concentration of 2,5-HD in urine. The probable effect of toluene on the concentration of 2,5-HD was also discussed in the present study.     

Evaluation of 2,5-hexanedione in urine of workers exposed to n-hexane in Brazilian shoe factories

Urinary 2,5-hexanedione (2,5-HD) is used as a biomarker for biological monitoring of workers exposed to n-hexane. The purpose of this study was to compare two types of treatment of urine samples during clean-up (with and without acidic hydrolysis) and to study the exposure situation of workers exposed to n-hexane during shoe manufacturing. There, various glues containing n-hexane are used. Quantification of 2,5-HD was carried out by gas chromatography and flame ionization detection (GC-FID). Fifty-two urine samples taken from workers of seven shoe factories were analyzed. Thirty-four persons from the administrative staff of the same factories served as controls. They were not known to be exposed to n-hexane. The samples treated with acidic hydrolysis showed levels (average 0.94 mg/l) approximately 10 times higher than samples without acidic hydrolysis (0.09 mg/l). The difference is predominantly caused by the conversion of other metabolites of n-hexane (e.g. 4,5-dihydroxy-2-hexanone) to 2,5-HD in the presence of acids. Our results also show, that exposure to n-hexane is different between various industries. Levels of 2,5-HD in urine are predominantly dependent on the type of operation (how the glue is applied on the leather during shoe manufacturing). Simple measures, e.g. using a glue handgun instead of a paintbrush significantly decreased exposure to n-hexane.     

Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.     

Uncoupling of Cerebral Glucose Supply and Utilization After Hexane-2,5-Dione Intoxication in the Rat

Chronic administration of hexane-2,5-dione (2,5-HD) to rats causes an accumulation of neurofilaments within axons that may lead to their degeneration. This occurs in both the CNS and PNS. It has been suggested that one of the effects of 2,5-HD is an impairment of glucose utilization arising from an inhibition of specific glycolytic enzymes. This hypothesis is based principally on evidence obtained in vitro. In the present study, glucose utilization, glucose transport across the blood-brain barrier, and blood flow have been measured in vivo in brain regions of control rats and in three groups of rats treated with 2,5-HD as (a) a single intragastric dose (500 mg/kg of body weight), (b) high chronic doses of 500 mg/kg of body weight for 15 days, or (c) low chronic doses of 250 mg/kg of body weight for 21 days. Group b showed overt signs of neuropathy, whereas groups a and c did not. The results indicate two independent effects of 2,5-HD in the CNS: a dose-dependent inhibition of glucose utilization and an effect on glucose supply and transport across the blood-brain barrier, which is apparent only after chronic treatment.     

In vitro binding of [14C]2,5-hexanedione to rat neuronal cytoskeletal proteins

2,5-Hexanedione (2,5-HD) induces central-peripheral axonpathy characterized by the accumulation of 10-nm neurofilaments proximal to the nodes of Ranvier and a Wallerian-type degeneration. It has been postulated that neurofilament crosslinking may be involved in the production of this axonopathy. A potential initiating event in this neurotoxic process may be the direct binding of 2,5-HD to neurofilament and microtubule proteins. In this study, the in vitro binding of [¹⁴C]2,5-HD to neurofilament and microtubule proteins was examined. Neurofilament proteins isolated from rat spinal cord or microtubule proteins isolated from rat brain were incubated in the presence of 2,5-HD at concentrations ranging 25 to 500 mM. Quantitative analysis of sodium dodecyl sulfate (SDS) polyacrylamide gels revealed a dose- and time-dependent binding of 2,5-HD to both neurofilament proteins and microtubule proteins. Expressed as pmol 2,5-HD bound per g protein, the observed relative binding was MAP2>NF160>NF200>NF68>tubulin. These data demonstrate the direct binding of 2,5-HD to cytoskeletal proteins including both neurofilaments and microtubules.     

Effect of 2,5-hexanedione and 3,4-dimethyl-2,5-hexanedione on retrograde axonal transport in sciatic nerve

The effects of systemically introduced neurotoxic solvents 2,5-hexanedione (2,5-HD) and 3,4-dimethyl-2,5-hexanedione (DMHD) on retrograde axonal transport (RT) of¹²⁵I-labeled tetanus toxin (TT) was studied in rat and mouse sciatic nerves. The rate of retrograde transport of TT in control rat sciatic nerves was slightly higher (6.8±0.4 mm/h) than in mouse sciatic nerves (5.4±0.5 mm/h). A single high dose of 2,5-HD (1,000 mg/kg, i.p.) produced a time-dependent effect on RT in mouse sciatic nerves. 2,5-HD caused a gradual decrease in the velocity of RT (approximately 65% inhibition between 2.0–2.5 h) with a reversal to normal rate 3–5 h after the toxin administration. The effect of DMHD on RT was examined following semi-chronic treatment in rats. DMHD caused a significant decrease (approximately 50%) in the rate of TT transport, in addition, it produced weight loss and hind-limb paralysis.I had the good opportunity of being a member of Professor Alan N. Davison' research team during 1971–1977. This research paper is dedicated to his retirement.     

Plasma endotoxin and concentrations of stable metabolites of prostacyclin, thromboxane A2, and prostaglandin E2 in postpartum dairy cows

The presence of endotoxin in plasma and patterns of stable metabolites of prostacyclin (PC), thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) were determined during the first postpartum estrous cycles in sixteen dairy cows. These included 8 cows with uterine infections which exhibited shortened luteal phases (SC) and 8 cows which had normal luteal phases (NC) after the first post partum ovulations. Endotoxin was consistently detected in all SC cows during the abbreviated estrous cycles while plasma samples of NC cows were free of endotoxin. Plasma concentrations of TXA2 metabolite was higher in SC cows (p less than 0.05) (1785-3452 pg/ml) compared to NC cows (723-1240 pg/ml). Similarly, plasma concentrations of PC metabolite was higher in SC cows (p less than 0.07) (423-1847 pg/ml) compared to NC cows (159-325 pg/ml). In contrast, plasma concentrations of PGE2 metabolite was higher in NC cows (p less than 0.05) (850-2219 pg/ml) compared to SC cows (455-628 pg/ml). The results of this study suggest that postpartum uterine infections mediate the release of prostaglandins from the uteri by means of the endotoxin and endotoxin appears to stimulate selectively the production of PC and TXA2 favoring early demise of corpora lutea formed after first postpartum ovulations in dairy cows.     

The blood pressure and renal responses to SCH 34826, a neutral metalloendopeptidase inhibitor, and C-ANF (4-23) in Doca-salt hypertensive rats.

C-ANF (4-23) and neutral metalloendopeptidase (NEP) inhibitors have been shown to prevent ANF metabolism and lower blood pressure presumably by the accumulation of ANF in the circulation. In the present study, we examined the interaction between C-ANF (4-23) and SCH 34826, an inhibitor of NEP, and ensuing effects on blood pressure, excretion of urine and sodium, and cGMP in the plasma and urine in conscious DOCA-salt hypertensive rats. C-ANF (100 micrograms/kg, iv bolus plus 10 micrograms/kg/min X 30) or SCH 34826 (90 mg/kg, sc) alone caused significant reductions in blood pressure and increases in plasma and urinary excretion of cGMP, a biochemical marker of endogenous ANF activity, at one hour post-drug. C-ANF (4-23) alone elicited a significant diuresis and natriuresis. SCH 34826 also enhanced sodium excretion and tended to increase urine volume. In comparison, the combination of C-ANF (4-23) and SCH 34826 produced a greater reduction in blood pressure and increases in plasma and urinary excretion of cGMP than either agent alone. The combination also caused significant diuresis and natriuresis. It is suggested that the greater blood pressure and renal responses to a combination of SCH 34826 and C-ANF than either agent alone reflect greater accumulation of endogenous ANF due to concomitant inhibition of both receptor-mediated clearance and NEP.     

The imbalance between dynamic and stable microtubules underlies neurodegeneration induced by 2,5-hexanedione

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of α-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration.     

Blood pressure dipping and salivary cortisol as markers of fatigue and sleep deprivation in staff anesthesiologists

Anesthesiologists often work extended duty shifts that result in acute and chronic sleep loss and circadian disruption. Stress caused by sleep deprivation, together with excessive workload could contribute to acute increases in blood pressure (BP) and sympathetic nervous system activity. Non-dipping pattern of BP is considered an additional risk factor for cardiovascular events and target organ damage. We hypothesized that there would be significant changes of cardiovascular parameters when comparing work on call during the 24-hour in-hospital shift (24-HD) versus ordinary working day (8-HD) combined with changes of dipping pattern and altered diurnal cortisol secretion, measured by salivary cortisol (SC). Following local Medical Ethics Committee approval, 12 out of 36 staff anesthesiologists (8 male, 4 female), 33-61 years old, participated in this study. Ambulatory BP monitor was used for noninvasive 24-hour ambulatory BP and heart rate (HR) monitoring. Each participant was monitored continuously during the 8-HD, as well as during the 24-HD. Saliva for analysis of cortisol levels was collected six times a day (at 8 am, 11 am, 2 pm, 5pm, 8pm, and 11 pm) both during 8-HD and on 24-HD. There was a significant decrease in number of diastolic dippers on call vs. diastolic dippers on ordinary working day (4/12 vs. 10/12, p=0.036), and non significant decrease of systolic dippers (3/12 vs. 7/12, p =0.214). There were no significant differences in SC values between 8-HD and 24-HD at all observed time points. However, the SC values measured during the night were markedly elevated on both days compared with reference values and the shapes of SC curves were altered. The lack of diastolic BP dipping could be more sensitive indicator of stress among staff anesthesiologists than systolic BP dipping. The shape of SC diurnal curve in terms of elevated night values could be another indicator of their chronic fatigue.     

Redox status of the testes and sperm of rats following exposure to 2,5-hexanedione

Objectives: Exposure to 2,5-hexanedione (2,5-HD) is well known to be associated with reproductive dysfunctions in both humans and animals. However, the role of oxidative stress in 2,5-HD-induced toxicity in testes and sperm has not yet been studied.

Methodology: The present study investigated the influence of 2,5-HD on antioxidant systems in the testes and epididymal sperm of rats following exposure to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 consecutive days.

Results: Administration of 0.5% 2,5-HD significantly (P?<?0.05) decreased epididymis weight, whereas 1% 2,5-HD-treated rats showed significantly decreased body weight, testis, and epididymis weights compared with the control group. Exposure to 2,5-HD caused a significant dose-dependent increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase in both testes and sperm compared with the control group. Moreover, 2,5-HD-exposed rats showed significant decrease in glutathione-S-transferase activity and glutathione level with concomitant significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm. Testicular and epididymal atrophy with significant, dose-dependent, decrease in epididymal sperm number, sperm motility, and viability were observed in 2,5-HD-treated rats.

Conclusion: 2,5-HD exposure impaired testicular function and sperm characteristics by disruption of the antioxidant systems and consequently, increased oxidative stress in the treated rats.     

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: A preliminary study

Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR = 5.58, 95% CI = 1.32-23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.     

Plasma endotoxin and concentrations of stable metabolites of prostacyclin, thromboxane A2, and prostaglandin E2 in postpartum dairy cows

The presence of endotoxin in plasma and patterns of stable metabolites of prostacyclin (PC), thromoxane A₂ (TXA₂) and prostaglandin E₂ (PGE₂) were determined during the first postpartum estrous cycles in sixteen dairy cows. These included 8 cows with uterine infections which exhibited shortened luteal phases (SC) and 8 cows which had normal luteal phases (NC) after the first post partum ovulations. Endotoxin was consistently detected in all SC cows during the abbreviated estrous cycles while plasma samples of NC cows were free of endotoxin. Plasma concentrations of TXA₂ metabolite was higher in SC cows (p<0.05) (1785–3452 pg/ml) compared to NC cows (723–1240 pg/ml). Similarly, plasma concentrations of PC metabolite was higher in SC cows (p<0.07) (423–1847 pg/ml) compared to NC cows (159–325 pg/ml). In contrast, plasma concentrations of PGE₂ metabolite was higher in NC cows (p<0.05) (850–2219 pg/ml) compared to SC cows (455–628 pg/ml). The results of this study suggest that postpartum uterine infections mediate the release of prostaglandins from the uteri by means of the endotoxin and endotoxin appears to stimulate selectively the production of PC and TXA₂ favoring early demise of corpora lutea formed after first postpartum ovulations in dairy cows.     

Comparison of nerve cell and nerve cell plus Schwann cell cultures, with particular emphasis on basal lamina and collagen formation

The availability of cultures of normal cells (NCs) and Schwann cells (SCs) with and without fibroblasts has allowed us to investigate the sources of endoneurial and perineurial constituents of peripheral nerve. NCs cultured alone, devoid of ensheathment but healthy in appearance, lack basal lamina and extracellular fibrils. In contrast, when SCs accompany NCs, basal lamina and extracellular fibrils are consistently visible around SCs in outgrowth areas formed de novo in culture. These fibrils average 18 nm in diameter, exhibit a repeating banding pattern, and are trypsin-resistant and collagenase-sensitive. Collagen synthesis is also indicated by the incorporation of [14C]proline into peptide-bound hydroxy-proline in NC + SC or SC cultures. That the [14C]hydroxyproline polypeptides formed in NC + SC cultures are collagenous was determined in part by pepsin digestion- ammonium sulfate precipitation-polyacrylamide gel electrophoresis techniques; the 14C-polypeptides migrate to the positions of alpha 1 (I), alpha 2, alpha 1 (III), and alpha B chains of type I, type III, and A-B collagens. Also formed are thin, ruthenium red-preserved strands interconnecting basal laminae. SC ensheathment of axons is similar to that found in the animal; one SC is related to a number of unmyelinated axons or a single myelinated axon. This proclivity to ensheathe and myelinate axons indicates that SC function is not lost during the preparative procedures or after lengthy isolation in culture and provides the most reliable means for SC identification. Perineurial ensheathment and macrophages are lacking in NC + SC culture preparations divested of fibroblasts. We conclude that SCs do not form perineurium or the larger diameter collagen fibrils typical of endoneurium but that in combination with neurons they generate biochemically detectable collagens and morphologically visible basal lamina and thin collagenous fibrils.     

The determination of isoniazid and its metabolites acetylisoniazid, monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine in serum and urine

1. A solvent system was devised for the extraction of isoniazid and its metabolites acetylisoniazid, monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine from serum and urine. 2. Specific chemical and fluorimetric methods were developed for the determination of the extracted isoniazid and acetylisoniazid, and chemical methods for the determination of monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine. 3. When applied to serum, these methods were capable of measuring concentrations of down to about 0.005mug of isoniazid/ml, 0.05mug of acetylisoniazid/ml, 0.2mug of monoacetylhydrazine/ml, 0.2mug of diacetylhydrazine/ml, 0.02mug of isonicotinic acid/ml and 0.1mug of isonicotinylglycine/ml. 4. In urine, these methods were capable of measuring concentrations of down to about 0.05mug of isoniazid/ml, 0.2mug of acetylisoniazid/ml, 1mug of diacetylhydrazine/ml, 0.1mug of isonicotinic acid/ml and 0.2mug of isonicotinylglycine/ml. 5. The stability of these compounds was studied in serum and urine and a method devised to decrease their decomposition in serum.     

Size-dependent Mating Behaviours of Male Sand-bubbler Crab,Scopimera globosa: Alternative Tactics in the Life History

Factors leading to the separation of mating behaviours were investigated in the sand-bubbler crab, Scopimera globosa. The crabs mated on the surface (surface copulation, SC) and underground (UC). UC males were large (old) whilst SC males were small (young). Burrowless females bred in the UC males' burrows. These females accepted UC in exchange for access to a burrow. UC occurred much more frequently than SC in the burrow area in which females oviposited. Most SC occurred in the water-saturated area affording a rich diet. SC was accepted by most large and small females in both areas and most UC by small females in the burrow area. SC was an alternative to UC for males in that there was a size dependence between types of copulation. These two mating behaviours involved different degrees of interaction with neighbouring males. Males attempting to carry a female to their burrows for UC were more often disturbed by other males than were males attempting SC. In the interaction for both UC and SC, larger males were likely to resist the disturbances. UC males needed their own burrows, but these burrows were not enlarged before mating. UC males have a higher paternity of eggs than SC males, because SC males' sperm is often displaced by other males. Thus, UC was a behaviour with relatively higher costs and benefits for male crabs than SC behaviour. Alternative mating behaviours in male S. globosa are conditional, and explained by intrasexual interactions and a male life history strategy with a trade-off between growth and reproduction. It is not likely that the size dependence of male mating behaviour is caused by mate preference of females for UC males in the burrow area.