Rapid microdetermination of D-glucose anomers with β-D-glucose dehydrogenase and mutarotase was deviced. The reaction mixture (1ml) composed of 20 units of purified β-D-glucose dehydrogenase (Bacillus cereus M 1020) and 2μmole of NAD⁺ dissolved in 20mM EDTA buffer (pH 6.5). By addition of D-glucose solution (5μl,<30μg of D-glucose) at 25°, arapid increase of absorption (NADH) at 340nm due to β-D-glucose was recorded, then 5units of mutarotase (hog kidney) was added in the reaction mixture to mutarotate the remaining α-D-glucose to β-D-glucose. The second increase of absorption (NADH) due to the α-D-glucose was recorded. From these two absorptions, the ratio of β to α-D-glucose was determined in 3.5-4min. This new method was not interfered by glutathione, cysteine, ascorbic acid, sodium fluoride, uric acid, etc. β-D-Glucose dehydrogenase from Gluconobacter oxydans can be used also for the same determination of D-glucose anomers, however, NADP should be used as a coenzyme

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Recovery rates for guanidino compound analyses were measured in ten laboratories. Authentic guanidino compounds were added to plasma, and plasma protein was removed by the ultrafiltration using Amicon Centriflo Membrane Cone Type CF 25 (Amicon Corp.). Guanidino compound analyses were performed by the guanidino compound autoanalyzer (model G-520 and GCA-101: Japan Spectroscpoic Co., Ltd. ; model JLC-6AH: Japan Electron Optics Lab. Co., Ltd. ; and other special types). When the samples were adjusted to pH 2-4, the recovery rate for each guanidino compound in plasma was rather high and stable. On the con trary, the recovery rates for taurocyamine, guanidinosuccinic acid, guanidinoacetic acid and β-guanidinopropionic acid were rather lower, when the pH of samples were not adjusted. Therefore the samples should be adjusted to acidic before ultrafiltration, for avoiding adsorption of guanidino compound on an ultrafiltration membrane.
Guanidino compound analyses of ECUM solution and plasma were carried out on the same samples in twelve laboratories. Guanidino compound analyses were performed by several kinds of different methods depend on liquid chromatography and fluoremetry. As the results of the experiment, we found that the variance of measured values by the differen laboratories was very large. This variance may be mainly caused on pretreatment for analysis.

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In the course of analysis; sample storage, deproteinization and sample charging on a column, the concentration of hydrogen ion makes various effects on the observed value of free guanidino compounds in plasma. The pH of plasma increases by storage at room temperature and at-20°. And GAA and MG also increase remarkably by storage at room temperature, but do not increase at-20°. G and MG increase when the plasma is placed in acidic condition before deproteinization by ultrafiltration. Ghost peaks appeare on the chromatogram when the sample was charged on the column in the basic condition, and acidic guanidino compounds show lower value.
GAA and MG should be produced during storage at room temperature, and G and MG should be released from plasma protein in the acidic condition. Acidic guanidino compounds should slip down from the stational phase and make some ghost peaks when the sample was charged in the basic condition.
It is important to be careful of storage condition of plasma and to keep a check on the concentration of hydrogen ion in every step of the analysis.

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The study on pretreatment in analysis of guanidinocompounds resulted in the following knowledges.
1) Ultrafiltration of acidified sample solution served for highest recovery.
2) Taking into accont rapidity of the measurement, it seems reasonable that the time taken to ultrafilter is about one hour.
3) It is assumed that the dilution of sample solution affect the concentration of free-guan-idino-compounds unbound to protein in plasma, and that this procedure change the permiability of guanidino-compounds through membrane.

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We have studied whether Guanidino compounds in sample are adsorpted on surface of polyethylene syringe or not in comparison with glass syringe.
We have used measured values of GSA, GAA and MG in sample, and a difference between values from polyethylene and glass syringe was not recognized.
We have also compared values oftained from serum and plasma, and have not recognized a difference between their values using freshly collected samples.

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We studied the advantage and reliability of the previously reported method and newly developed automated method for the determination of Methylguanidine (MG) and Guanidinosuccinic acid (GSA) which are prime candidates for uremic toxin.
The same samples obtained from hemodialized patients were analyzed for MG and GSA concentration by the previouly rerorted method and/or automated method in 13 institutes similtaneously, and the results were compared. In order to evaluate the column chromatographic performance of the both method, MG and GSA fraction of uremic serum which waseluted by the previously reported method first was then aplied to elute by automatic guanidine analyzer, respectively.
The previouly reported method using fluorometry was sensitive, easy, economical and yielded good results on the isolation of MG and GSA from uremic serum, so it was considered that this method was suitable for rutine use in clinical practice. On the other hand, the results of MG and GSA determination by automated method showed significant difference among each institute especially in plasma concentration. However this method requires only small amount of sample and is useful in similtaneuos determination of several other guanidino compounds, so it should be hoped that this method will be made improvement and developed furthermore.

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A new fluorimetric method for the determination of monosubstituted guanidino compounds with benzoin is described. This is based on that fluorescence develops when the guanidino compound is heated in aqueous potassium hydroxide solution with benzoin in the presence of dimethylformamide, and the flu°rescence produced is stabilized by β-mercaptoethanol. The reaction conditions of the method were established by employing methylguanidine and arginine as model compounds. The prescribed method requires the heating time of 45min at 100°, and the fluorescence produced shows excitation and emission maxima around 325 and 435nm, respectively. The method is simple, selective for monosubstituted guanidino compounds including polypeptides with one or two arginyl residues, and sensitive (thelower limits of determination, 0.08-0.22nmol/ml).
Then, the method was revised for a rapid and a more sensitive method, based on the findings that the reaction mixture fluoresced most intensely when made weakly alkaline (pH, around 9) after heating for only several min. A mixture of sodium dihydrogen phosphate and Tris for the pH adjustment and 7min of the heating were employed in the revised method. The fluorescence from the guanidino compounds shows excitation maximum around 322 and emission maximum around 440nm. The rivised method is sensitive (the lower limits of determination, 0.09-0.17 and 0.04-0.06 nmol/ml for the compounds with one and two arginyl residues, respectively), precise (CV, 1.8% for 10nmol/m/ arginine, n=30), and selective (no interfering compounds of biological importance). The method should be applicable to the asssy of the guanidino compounds in biological samples, which are implicated as uremic toxins, after the compounds are separated (from the sample) by liquid chromatography.

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Since the elevated level of guanidinosuccinic acid was demonstrated in sera of uremic patient by Bonas et al.(1963), so many studies have been focused on analyses of gua-nidino compounds as uremic toxins. In this monograph, more than 50 of naturally occurring guanidino compounds were classified, and analytical methods for guanidino compounds were reviewed compactly as follows:
I. Classification of naturally occurring guanidino compounds
1. Guanidine and its derivatives
·guanidine ·methylguanidine ·dimethylguanidine ·hydroxyguanidine
2. ω-Guanidino acids and its derivatives
·guanidinoacetic acid (glycocyamine) ·β-guanidinopropionic acid ·γ-guanidinobutyric acid ·γ-guanidinobutyramide ·δ-guanidinovaleric acid ·creatine ·asterubine ·argininic acid (α-hydroxy-δ-guanidinovaleric acid) ·α-keto--guanidinovaleric acid
3. Acidic guanidino acid
·guanidinosuccinic acid ·α-guanidinoglutaric acid
4. Arginine and its derivatives
·arginine ·α-N-acetylarginine ·agmatine ·methylagmatine ·δ-N-hydroxyarginine ·acetylagmatine ·γ-hydroxyarginine ·α-N-acetyl-r-hydroxyarginine ·NG, NG-dimethylarginine ·NG, N'G-dimethylarginine ·octopine ·arginosuccinic acid ·canavaninosuccinic acid ·suberylarginine ·homoarginine
5. Guan'dinoethane derivatives
·taurocyamine (guanidinoethylsulfonic acid) ·hypotaurocyamine (guanidinoethylsulfinic acid) ·guanidinoethylmethylphosphate (opheline) ·guanidinoethylserylphosphate (lombricine) ·guanidinoethyl-(α-N, N-dimethyl) serylphosphate (thalassemine) ·guanidinoethylaspartylserylphosphate (aspartyllombricine) ·asterubine
6. Diguanidino compounds
·arcaine (diamidinoputrescine) ·audouine (diamidinocadaverine) ·hirudonine (diamidinospermidine) ·vitiatin
7. Phosphagenes
·argininephosphate ·creatinephosphate ·glycocyaminephosphate ·taurocyaminephosphate ·hypotaurocyaminephosphate ·ophelinephosphate ·lombricinephosphate ·thalasseminephosphate
8. Others
·galegine ·phascoline ·phascolosomine ·canavanine ·streptidine ·creatinine
II. Analytical methods for guanidino compounds
1. paper chromatography and thin layer chromotography
2. liquid column chromatography
3. gas chromatography-mass spectrometry

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