Improved Gas Chromatographic Determination of Guanidino Compounds Using Isovaleroylacetone and Ethyl Chloroformate as Derivatizing Reagents.

An improved GC method in terms of sensitivity and decrease in the analysis time has been developed for the analysis of eight guanidino compounds: guanidine (G), methylguanidine (MG), creatinine (CTN), guanidinoacetic acid (GAA), guanidinobutyric acid (GBA), guanidinopropionic acid (GPA), argenine (Arg), and guanidinosuccinic acid (GSA), using isovaleroylacetone (IVA) and ethyl chloroformate (ECF) as derivatizing reagents. The separation was obtained from column HP-5 (30 m × 0.32 mm i.d.) with film thickness of 0.25 µm within 11 min. The linear calibrations were obtained with 0.5 to 50 µg/mL with coefficient of determination (R(2)) within 0.9969 - 0.9998. Limits of detections (LODs) were within 5 - 140 ng/mL. The derivatization, separation and determination was repeatable (n = 6) with relative standard deviation (RSD) within 1.2 - 3.1%. The guanidino compounds were determined in deproteinized serum of healthy volunteers and uremic patients within below LOD to 8.8 µg/mL and below LOD to 43.99 µg/mL with RSD within 1.4 - 3.6%. The recovery of guanidino compounds calculated by standard addition from serum was within 96.1 - 98.9%, with RSD 1.4 - 3.6%.

Sustained increase of methylguanidine in rats after amygdala or hippocampal kindling.

Methylguanidine (MG) and guanidinoacetic acid (GAA) are known to be endogenous convulsants in the mammalian brain. We have reported previously that MG and GAA concentrations were significantly elevated bilaterally in the amygdala (AM) 7 and 28 days after completion of AM kindling. In the present study, we investigated regional changes in MG and GAA in (1) AM-kindled rat brain 3 months after completion of kindling and (2) hippocampal (HIPP)-kindled rat brain 7 and 28 days after completion of kindling, using high-performance liquid chromatography. MG was increased significantly in the kindled AM 3 months after completion of AM kindling (76%, P < 0.05). Significant increases in MG levels were also found 7 and 28 days after completion of HIPP kindling (124%, P < 0.025 and 110%, P < 0.05, respectively) in the left AM, ipsilateral to the kindled site. GAA increased significantly (93%, P < 0.025) only in the left AM, ipsilateral to the kindled site, 7 days after completion of HIPP kindling. These results, together with our previous data, indicate that long-lasting increases of MG occur in the AM regardless of kindled foci, and may be important in producing neuronal hyperexcitability in kindled epileptogenesis.

Activity of (-)-epigallocatechin 3-O-gallate against oxidative stress in rats with adenine-induced renal failure.

Methylguanidine (MG) is widely recognized as a strong uremic toxin. The hydroxyl radical (*OH) specifically plays an important role in the pathway of MG production from creatinine (Cr). In this study, we investigated whether oral administration of (-)-epigallocatechin 3-O-gallate (EGCg) suppresses MG production in rats with chronic renal failure after intraperitoneal Cr injection. MG production from Cr was significantly increased in rats with adenine-induced renal failure, which was more vulnerable to oxidative stress, compared with that in normal rats. However, oral administration of EGCg 30 min before and after Cr injection effectively inhibited MG production. Our findings suggest that EGCg, an excellent antioxidant from green tea, exerts protective activity in rats with chronic renal failure, resulting in suppression of Cr oxidation influenced by *OH.

Methylguanidine content in food.

A combination of column chromatography using microcrystalline cellulose and ion-exchange resin, paper chromatography, gas chromatography, and gas chromatogrraphy-mass spectometry was used to determine the content of methylguanidine in various meats of fish and animals, and their processed products. All the samples of fresh fish meat and beef analyzed contained trace amounts (0.2 approximately 0.9 ppm) of methylguanidine. Various types of dried or canned fish also contained trace amounts, while most of the "Bushi-rui," a highly processed dried meat of bonito, mackerel, or sardine contained 21 to 106 ppm of the compound. Two brands of peptone made by digesting meat probably with pepsin and trypsin also contained 12 and 70 ppm of methylguanidine, respectively. Samples of ham, sausage, salami sausage, powdered skim-milk, and a few others did not contain any detectable amount (less than 0.2 ppm).

Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography-fluorescence detection method.

A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These quanidino compounds are separated on a 6 x 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.

Determination of methylguanidine in plasma and urine by high-performance liquid chromatography with fluorescence detection following postcolumn derivatization.

A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure.

Gas chromatographic determination of methylguanidine, guanidine and agmatine as their hexafluoroacetyletonates.

Seven derivatives of each of methylguanidine, guanidine and agmatine have been prepared, and the specificity and volatility of their gas chromatographic detection have been studied. The hexafluoroacetylacetonates have been found to be the most specific for the three guanidines, and are highly sensitive to alkali flame ionization and electron-capture detections. These derivatives are also fairly resistant to hydrolysis occurring in the derivatization process.

Comparison of dialysis programmes on different molecular prescriptions: a preliminary study.

Five dialysis and/or adsorbent therapy programmes were compared to examine the biological toxicity of both small and middle molecular fractions from the clinical viewpoint. With combination of various surface areas, diffusion pool sizes, or use of charcoal, what can be called a 'molecular prescription' is possible. The programmes were designed to bring about different degrees of removal of both fractions. Ultrafiltrates obtained with Amicon XM-10 hollow fibre filters before and after a programme were examined for levels of BUN, creatinine, and methylguanidine, inhibitory effect on pyruvate kinase, G-6-PDH, and LDH, and on the cell mortality rate of rat embryo liver cell monolayer culture. The worst results were obtained in those programmes which retained small molecular fractions and removed the middle molecular fractions efficiently. The biological toxicity of the small molecular fraction, from the viewpoint of maintaining the 'milieu intérieur', seems to be much stronger than that of the middle molecular fraction. A dialysis or adsorbent therapy programme removing the middle molecular fraction only, but leaving the small molecular fraction should be considered as putting the cart before the horse.

Occurrence of methylguanidine and agmatine in foods.

Methylguanidine (MG), a precursor of carcinogenic methylnitrosocyanamide and methylnitrosourea, has long been considered to occur widely in fresh beef and various fish muscles in fairly high concentrations. Agmatine (AG) has been reported to be easily nitrosated to give a potent mutagen under acidic conditions. A survey has been conducted on the MG and AG contents of various fresh and processed foods. The present study reveals that no appreciable amount, or only trace amounts, of MG could be detected in fresh beef, pork, chicken and various fish and shellfish. In addition, almost the same low levels of MG were detected in various processed foods, except in the case of smoked-dried fish products, where the MG values ranged from 18 to 178 mg/kg. Relatively high concentrations of AG, ranging from 40 to 200 mg/kg, could be detected in fresh abalone and top-shell muscles, while fairly high concentrations of AG could be detected in some processed foods. A dried squid product, in particular, was found to contain as high as 650 mg/kg of AG.

Factors affecting the metabolic production of methylguanidine.

1. Methylguanidine administered orally to normal volunteers was almost completely recovered in the urine, indicating that it is absorbed in the gastrointestinal tract and is not converted into other compounds. In normal persons at least, its urinary output therefore corresponds to its metabolic production rate plus the amount ingested. 2. In normal persons, diets based on foods not containing methylguanidine (e.g. vegetarian, protein-free and milk-egg) caused a fall in the urinary output of methylguanidine as compared with the output of the same subjects on a free diet. Conversely, higher amounts of methylguanidine were excreted on a diet rich in broth and in boiled beef, which contain large amounts of methylguanidine formed from the oxidation of creatinine, caused by boiling. 3. Oral administration of creatinine to normal volunteers induced an immediate and marked increase in urinary excretion of methylguanidine, and the ingestion of [methyl-14-C]creatinine by uraemic patients was followed by the urinary excretion of labelled methylguanidine. These findings indicate that creatinine is partly converted into methylguanidine in both normal and uraemic subjects and accounts for the high metabolic production of methylguanidine in patients with renal failure, in whom the body pool of creatinine is high. 4. Creatinine, incubated at 38 degrees C for 24 h in Krebs bicarbonate solution (pH 7-38) through which was bubbled oxygen with 15% carbon dioxide, was partially oxidized to methylguanidine. This raises the possibility that even in vivo such a conversion may occur "non-enzymatically".

Production of methylguanidine from creatinine in normal rats and rats with renal failure.

Creatinine (Cr) was administered intraperitoneally to both normal rats and those given adenine, and time-course changes in methylguanidine (MG) production from Cr were compared. In rats with renal failure, the accumulation of MG in the body increased gradually with time after Cr administration. In particular, the MG level in skeletal muscle was markedly high in comparison with that in serum, liver or kidney, and a high concentration of MG was still present 24 h after Cr loading. In contrast, the amount of MG excreted into urine in these rats during 24 h after Cr administration was lower than the corresponding values in normal rats. Thus, the amount of MG per rat, distributed at 24 h after intraperitoneal administration of Cr 300 mg/100 g body weight was calculated. The production of MG from Cr was found to be markedly higher in rats with adenine-induced renal failure (172.09 micrograms/100 g body weight) than in normal rats (70.30 micrograms/100 g body weight). Produced MG was mostly excreted into urine in normal rats, whereas in rats with renal failure as much as 79.1% of produced MG was accumulated in the body.

Increase of methylguanidine and guanidinoacetic acid in the brain of amygdala-kindled rats.

Guanidino compounds are intrinsic chemoconvulsants. We investigated the regional and time-dependent changes of these compounds in the amygdala (AM) of kindled rats versus electroconvulsive shock (ECS) seizures using high-performance liquid chromatography (HPLC). Twenty-eight days after the last AM-kindled seizure, guanidinoacetic acid (GAA) and methylguanidine (MG) levels were significantly increased in the bilateral AM as compared with those of control rats, which had been implanted with electrodes but were not stimulated. Both compounds, however, tended to decrease in the bilateral AM after ECS seizure. In addition, we measured these compounds after induction of one afterdischarge (AD) in the AM. These compounds increased significantly 7 days after AD was induced in the ipsilateral AM. We suggest that the increase of these compounds is coincident with AD generation and specific to AM kindling and the kindled state.