It is necessary for the diagnosis of uremia to determine the blood urea (U), uric acid (UA), creatinine (Cr) and methylguanidine (MG), which are uremic toxins, and for judging dialysance to determine vitamine B₁₂ (B₁₂). The simultaneous determination of these substances by high performance liquid chromatography (HPLC) was not reported because it was difficult to separate these substances one another and to separate these substances, especially U, from admixtures in the serum. Proposed method to separate these substances by paired-ion reverse phase HPLC was as follows ; (1) to determine U and UA in serum column ; Zorbax ODS, mobile phase ; phosphate buffer (con-taining 5 mM sodium phosphate, pH 2), to which 2.5 mM sodium hexane sulfate was added as counter ion (2) to determine UA, Cr and MG column and mobile phase were the same as described in (1) except that 80 mM sodium perchlorate was added to the mobile phase (3) to determine Cr, MG and B₁₂ column ; Nippon bunko ODS SS-10A, mobile phase ; perchlorate buffer (containing 15 mM sodium perchlorate, pH 5), to which 20% (v/v) acetonitrile was added. Column temperature was ambient and injection volume was 10μl in the method of (1), (2) and (3). Detection wave length and detection limit of these substances were U : 210 nm, 500 ng, UA : 234, 280 nm, 0.5 ng, Cr : 234 nm, 10 ng, MG : 210 nm, 100 ng and B₁₂ 210 nm, 10 ng, respectively. Serum urea determination by reverse phase HPLC without any disturbance of admixtures in the serum was achieved successfully for the first time by use of this method.

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A flow injection method for determining cyanide was applied to the screening of waste water samples from metal plating processes. A sample flowing at 1.2 ml/min was mixed with a sulfuric acid solution stream [2% (v/v), 0.4 ml/min] to adjust its pH to below 2. The acidified solution (200 μl) was introduced into a carrier stream [10% (w/v) NaCl, 2.0 ml/min] via an injector valve. Hydrogen cyanide liberated was diffused into a collector stream (0.1 N NaOH, 1.2 ml/min) through a Teflon membrane in a gas diffusion cell at 40°C. The collector solution was monitored with a flow-through cyanide ion selective electrode. The apparent permeability of cyanide was calculated as 34% at the 1 μg/ml level. A linear relationship was observed between the peak height and logarithmic concentration of cyanide in the range of 0.3-100μg/ml. The values determined by the present method (x) were in fair agreement with those obtained by the official method (y) for cyanide liberated at pH5. The following correlation was obtained between the two methods ; y=0.957 x+0.013 (r=0.946, n=50). By this method, 40 samples can be analyzed in one hour.

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A method for the quantitative analysis of sympathomimetic amines, methamphetamine (MA) and amphetamine (A), in the urine was developed by using column extraction-high-performance liquid chromatography (HPLC). Simple and rapid extraction of MA and A from the urine samples was carried out by using Bond Elut^[○!R] pre-packed column with acidified acetonitrile aqueous solution. The mean extraction ratio was 94.9% with the coefficient of variation of 1.66% (n=5). The HPLC was performed by utilizing a reversed-phase column with 0.05M ammonium acetate and 0.2% acetic acid in 50% acetonitrile and by monitoring at 254 nm. The sympathomimetic amines were eluted at a flow rate of 1.0 ml/min and the quantity of MA was estimated from the peak height. This method facilitated MA determination within 1.69% error over the urine level range from 5 to 100 μg/ml, with the limit of detection being 1 μg/ml. After intravenous injection of 2 mg MA in rats, the mean amount of MA excreted after 24 h in the urine was about 25% of the dose administered. The method was successfully applied to human urine samples containing MA.

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Attempts were made to reveal the composition of paralytic shellfish poisons (PSP) in the mussel Mytilus edulis from Funka Bay, Hokkaido. The shucked mussels were extracted with 80% ethanol (pH 2.0). The extract was defatted with chloroform, treated with activated charcoal, and then purified by column chromatography on Bio-Gel P-2 and Bio-Rex 70. Each fraction thus obtained was analyzed by fluorescence intensity monitoring and mouse assay. PSP components contained in toxic fractions were identified by using cellulose acetate membrane electrophoresis and thin layer chromatography on silica gel. The mussel from Funka Bay was found to contain gonyautoxin VIII and its epimer, together with gonyautoxin I-V, saxitoxin and neosaxitoxin. The ratios of each PSP component in the total toxicity were as follows ; gonyautoxin I (31%), gonyautoxin II (12%), gonyautoxin III (15%), gonyautoxin IV (16%), gonyautoxin V (3%), gonyautoxin VIII plus its epimer (2%), saxitoxin plus neosaxitoxin (21%). This is the first time that gonyautoxin VIII and its epimer, which have been mainly detected in the bivalves from the western part of Japan, are detected in Hokkaido. It was revealed from these results that low toxic components having N-sulfonato carbamoyl group such as gonyautoxin V and VIII are present not only in the western part of Japan but also in the eastern part of Japan, particularly in Hokkaido.

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Methamphetamine and its metabolites in hair samples obtained from habitual user of methamphetamine were identified and determined by mass fragmentography (MF). Hair sample was washed with water and methanol. The hair was crushed in 0.6 N HCl, suspension was alkalized with Na₂CO₃, and extracted with chloroform-isopropanol (3 : 1, v/v). The extract obtained was trifluoroacetylated, and applied to MF. Methamphetamine was detected in all hair samples obtained from 9 habitual users, and its contents were 0.7 to 67.5 ng/mg hair. Amphetamine, which was known as a metabolite of methamphetamine, was also detected in 6 hair samples of 9. Methamphetamine could be detected in hair sample obtained from the 37 d drug-free abuser. It is considered that analysis of methamphetamine in hair samples by MF is one of useful methods for discrimination of habitual user of methamphetamine. Furthermore, distribution of methamphetamine to top, middle and root parts of hair was examined. The results suggest that the distribution of mathamphetamine in a hair may be correlated with the individual history of abuse of drug.

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The mutagenic activities of diesel exhaust particles were examined by means of the Ames test. Two types of diesel vehicles (passenger car and truck) were operated on a chassis dynamometer at constant speeds (idling, 20, 40, 60 and 80 km/h) and variable speeds (the ten-mode pattern, the M-15 mode and typical traffic patterns in Tokyo). The exhaust particles were collected on quartz glass fiber filters, which were then extracted with benzene-ethanol mixture (4 : 1, v/v). Each extract was tested by the Ames Salmonella assay. The exhaust particle extracts of both vehicles showed high mutagenic activities on TA 98 and TA 100 strains with and without metabolic activation. The mutagenic activity per km at constant speed driving increased with increase of the speed. Furthermore, acceleration and deceleration caused increases of mutagenic activities per km. The mutagenic activities of the exhausts of both vehicles were positively correlated with particle emission but not with the main gaseous emissions (nitrogen oxides, hydrocarbons, carbon monoxide and carbon dioxide).

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The mutagenicity of chlorine-treated phenylphenols (o-, m-, and p-isomers) solutions was tested by the Ames procedure with Salmonella typhimurium TA 98 and TA 100 in the absence and presence of rat liver homogenate (S 9 mix). The Ames tests showed that chlorination of these compounds with an excess of chlorine in water leads to the formation of lipid-soluble and nonvolatile mutagens (positive in strain TA 100 without S 9 mix). Ether extracts of the aqueous p-phenylphenol solutions treated with low chlorine doses were also mutagenic and toxic in both strains TA 98 and TA 100 without S 9 mix. The strongest mutagenic and toxic effects on strain TA 100 without S 9 mix were observed with extracts of the aqueous solutions treated with chlorine under acidic conditions, and there was a pattern of decreasing mutagenicity with increasing pH. In addition, it was found that the mutagenic substances produced in the reactions can be removed from the chlorinated aqueous solutions by activated carbon.

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Elutions of vinyl chloride monomer (VCM) from polyvinyl chloride (PVC) resin pipes in the absence and presence of chlorine in water were compared by the method of head space analysis. The VCM concentrations in the absence of chlorine in water after contact with PVC pipes were dependent on the residual VCM contents in these pipes. In the presence of chlorine, however, lower concentrations of VCM in comparison with those values obtained in the absence of chlorine were observed for all PVC pipes examined. The decrease in both VCM and chlorine concentrations were also recognized when these chemicals were mixed in water, especially under acidic conditions. Gas chromatography-mass spectrometry of ether extract from the aqueous solution showed the reaction product of VCM and chlorine to be 2, 2-dichloroethanol.

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Environmental toxicology of metals, like that of other chemicals, consists of ecotoxicology and biotoxicology. Among the biotoxicology, pathophysiological approach, along with toxicological approach, is of particular importance for understanding effects of metals on human health and establishing measures to cope with them including biological monitoring and development of exposure parameters and preventive methods. Experiments have been carride out on chemical biology of chromium and clinical biochemistry of organic tin compounds. The aim of these studies is to demonstrate and evaluate the pathophysiological approach in the study of metal toxicity. Recent results in our laboratory are as follows. 1. Chemical toxicology of chromium. Two low-molecular-weight chromium-binding substances, LMCrI and II, were purified from the liver of dogs. Chromium bound to them showed a lower acute toxicity and higher rates of urinary excretion and renal clearance than potassium dichromate (VI) and chromium (III) chloride. The results together with other findings indicate that LMCrs play an important role in the detoxification and excretion of chromium in mammals. They also show that chemical form of metals and binding protein in the body are important factors in metal toxicity. 2. Clinical biochemistry of organic tin compounds Triphenyltin and tributyltin have been shown to inhibit insulin release from pancreatic islets, subsequently inducing diabetic lipemia due to insulin deficiency. The organic tins also inhibited collagen-induced aggregation and ATP secretion of platelets. Furthermore, they showed a dosedependent inhibition of O₂^- production, O₂ consumption and degranulation of leukocytes. These results indicate that organic tins affect a common mechanism of the stumulus-response coupling in the cell membrane.

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In the recent two decades, interest has been rising in metal-metal interaction in body. And at present the interactions to find its phenomenon is now being investigated. Therefore in the mechanism of interaction between metals, the hypothesis to explain this mechanism draw only out from these results reported. From these results, the following mechanism should be able to be indicated. (1) Competition in active site. There are competitions between metals against the activated substance or active site in body and those against absorption from a digestive tract, for example, the competition of lead and zinc to ALAD or the suppression by cadmium to the absorption of copper. (2) Formation of complex by metals. When two metals are administered simultaneously, the complex containing these metals is formed in body, and consequently metal toxicity is alleviated. This exsample is investigated in detail in Hg-Se interaction. (3) Induction of metal binding protein. This example can be seen in the induction of metallothionein. A metal which has the ability to induce Zn-thionein is used as pretreatment, and a metal which can replace Zn in Zn-thionein is used as challenge, and the toxicity of challenge metal may be mitigated.

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Urinary total protein, β₂-microglobulin, N-acetyl-β-D-glucosaminidase activity, metallothionein and cadmium were estimated in cadmium-exposed residents and control people. There was a close relationship between urinary β₂-microglobulin concentrations and results of proximal renal tubular function tests (uric acid clearance, % TRP, renal glucosuria, generalized aminoaciduria and low molecular weight proteinuria). It was concluded that urinary β₂-microglobulin concentration was a excellent index of proximal renal tubular dysfunctions. Residents' urinary β₂-microglobulin concentrations had a close relationship with urinary cadmium concentrations, indicating Dose-Effect Relationship between cadmium exposure and its health effect (i. e, proximal renal tubular dysfunctions). The increase of N-acetyl-β-D-glucosamidase activity in urine was recognized in cadmium-exposed residents. The increase of NAG activity might appear earlier than the increase of β₂-microglobulin in urine.

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The biochemical approach in analysis of heavy metals toxicity is described in this review. Especially, the toxic actions of tin and zinc, essential metals, are introduced. Tin has some toxic effects on gastrointestinal function, blood cells, hepatic enzymes, endocrine secretion from pancreas, renal calcium metabolism and its related enzymes, bone metabolism, central nervous system, and reproduction function. The toxic studies of inorganic tin on various biochemical indices, however, suggest that the critical organ of tin is bone with a decrease in the femoral calcium content. Zinc has a wide variety of roles in mammalian system. However, the dose of zinc, which stimulates the mitochondrial function in liver cells, nervous function, and the growth and calcification of bone, exerts a porotic effect in bone. This effect on bone results from the decrease in serum calcium levels. On the basis of the sign of this manifestation, the toxic effect level of zinc is estimated to be lower than 1.0 mg Zn/kg. On the other hand, lead, cadmium, aluminium, manganease, and lithium also cause a disorder of calcium metabolism and its related bone metabolism. Accordingly, this review emphasizes that alteration of serum calcium level is important for the manifestation of toxicity of metals in various biochemical indices.

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