Mechanism of Cypridina bioluminescence differs from that of firefly bioluminescence in that (1) the latter involves an activating step and (2) it shows a product inhibition. The chemical process of Cypridina luciferin producing light is discussed. Analytical application of a Cypridina luciferin analog is presented.

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Highly sensitive enzyme immunoassays using chemiluminescence reaction have been developed. Horseradish peroxidase and glucose oxidase were used as the labeling enzyme and conjugated with cortisol, dehydroepiandrosterone and its sulfate, 17α-hydroxyprogesterone, thyroxine, α-fetoprotein and insulin, respectively. Free and bound fractions after immune reaction, were separated by insoliblized antibodies or secondary antibodies.
The enxyme activities were measured by chemiluminescence reaction using luminol and hydrogen peroxide as substrates for horseradish peroxidase, and the glucose oxidase activity was also measured by luminol and potasium ferricyanide system/or trichlorophenol oxalate-fluorescence dye system after incubation with glucose. The faint chemiluminescence was measured by a photon counter or flow injection analysis system. Comparison of assay results obtained by radioimmunoassay and enzymeimmunoassay showed excellent agreement of results in all cases.
The detection limits of each substances were about 10^-15-10^-17 mol/assay tube. These enzymeimmunoassay based on chemiluminescence reaction were applicable to the routine determination of clinically important samples.

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Application of enzymes as an analytical tools has recently been an exponential increase particularly in the field of clinical chemistry. Among them, of special interest and importance is the use of immobilized enzymes, which offer several advantages over conventional methods with soluble enzymes: thermal stability, resistance to decomposition and ease to assemble in an automatic instrument.
On the other hand, many clinically important compound in blood are oxidized by an individual oxidase with concomitant formation of hydrogen peroxide. For the detection of enzymically generated hydrogen peroxide, luminol chemiluminescence method is the most favorable because of its high sensitivity, wide linear dynamic range and simple instrumentation. Furthermore the reagents are relatively inexpensive and stable, therefore, this enable us to couple the enzymic process and the chemiluminescence detection process through the continuous flow system. This is very important because the use of an immobilized enzyme column is probably only one possible way to perform the enzyme catalyzed process in the continuous flow system.
As the first step toward the development of an automatic multichannel enzymic analyzer, we have assembled an apparatus which enable us to analyze glucose and uric acid in blood sample with high sensitivity. The appratus consists of immobilized oxidase column and chemiluminescence detector. All reagents, luminol, ferricyanide and buffer solutions, are supplied continuously by a peristaltic pump, and blood samples are introduced into enzyme column from an autosampler or injected by microsyringe. In the enzyme column, glucose oxidase or uricase, which are immobilized onto long chain alkyl amine glass by the cyanogen bromide method, are packed.
Enzymically formed hydrogen peroxide are mixed with chemiluminescence reagents in a swirling cell mounted directly on the surface of window of photomultiplier.
This apparatus has been found to be successfully applied for the quantitative determination of glucose and uric acid in blood samples in a range between 10_-8 and 10_-4 M.
By slight modification of the apparatus NAD (P) H was also determined using methylene blue as an electron carrier between NAD (P) H and oxygen. This system responded to the concentration of NAD (P) H down to 10_-8 M.

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A chemiluminescence method for assay of polyamines in urine has been developed. Hydrolysis of conjugated polyamines in urine and extraction of polyamines from urine samples were carried out in a similar way to that reported by Okada et al.(Japan. J. Biochem., 53, 792 (1981)), as following.
0.5 ml of urine was treated with 20 U of acylpolyamine amidohydrase. After centrifugation the supernatant was applied to a Bio-Rex 70 column, which had been equilibrated with a 0.1 M tris buffer, pH 9.5 previously. Using 0.2 M HC1 as an eluant, polyamines were recovered complately in the eluate. The pH of the eluate was adjusted to 8.8 with 0.5 M NaOH and a 0.5 M tris buffer.
An aliquot of the eluate was used for polyamine assay by a chemiluminescence reaction.
The principle of the chemiluminescence reaction is as follows:
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_??_It took about 120 mm to complete the whole procedures from hydrolysis of conjugated polyamines to the measurement of the intensity of luminescence. 40 samples could be assayed within a working day. Polyamine concentrations in a range of 1 to 600μM could be determined by this method without dilution of samples. The recovery of putrescine, cadaverine, spermidine, and spermine were 100%, 111%, 89%, and 44%, respectively by this method. The coefficient of variation between runs (10 runs) were 4.4% for a sample with 28.3μM of putrescine, and 2.5% for a sample with 58.0μM of putrescine.
The concentration of the total polyamines in 24 hour urine samples collected from 51 healthy subjects (29 female and 22 male) was 36.2+23.5 μmol/g of creatinine, mean+2SD, and that in random fresh urine samples collected from 44 persons of the same group was 32.8+24.8μmol/g of creatinine.

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Chemiluminescence generated with the reaction of bis (2, 4, 6,-trichlorophenyl) oxalate and hydrogen peroxide was applied to a detection system for HPLC to determine fluorescamine-labeled catecholamines. The sensitivity of the chemiluminescence detection system was 25fmol for fluorescamine-labeled dopamine. Norepinephrine and dopamine in human urine were determined by the use of the new HPLC detection system with the coefficient of variation of less than 4.0%. Good correlations were obtained between the values for both norepinephrine and dopamine by the present method and the convetional fluorescence method.
In order to extend the utility of the new method we are tryin g to apply it to the determination of plasma catecholamines. However, for the determination of them in plasma, more sensitivity is required. For the purpose, we replaced the photomultiplier to a more sensitive one, changed the shape of a flow cell to a spiral type and re-investigated chemiluminescence reaction conditions from the view point of time courses of the reaction.

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The detecting ability of the photon counting system of single photoelectron counting method has been greatly improved and an equipment of this method, designed for biochemical and biomedical applications, was developed. The photomultiplier used in the instrument was of bialkali photocathode of 5cm diameter with the spectral responce nearly from 300nm to 660nm and of specifically low noise selected from hundreds of the same photomultipliers. The photomultiplier was cooled down to -20° in the equipment in the actual photon counting for the further reduction of the noise. All other possible electrical noises from the inside and the outside of the counting machine were eliminated or minimized. The sensitivity of the photon counting equipment reached the level high enough to permit the determination of extremely weak light emission like those of human blood and tissue samples. Thus, the first quantitative measurement, to our knowledge, of ultra weak chemiluminescence of human blood samples was performed with this instrument. The whole blood and plasma samples of normal subjects gave relatively low levels of light emission. On the contrary, the blood samples of patients with diabetes mellitus and with liver diseases showed significantly higher light emission levels. Of particular interest was of the blood of patients with obstructive jaundice and of severe hepatitis. The light emission of the blood samples of these disease was generally very high and in the levels allowing the spectral analysis of the emitted light.
The analysis and the scavenger experiments indicated the contribution of singlet oxygen as the photon emitting entity in these blood samples.

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Polyunsaturated fatty acid compositions were analyzed using gas liquid chromatograph (Shimazu, GC-R1A with solventless injector) equipped with a hydrogen flame ionization detector. A standard mixture of fatty acid was purchased from Applied Science, Inc. Two types of columns were used for the present measurement. One was a 300cm glass coiled column, 3mm i. d., packed with liquid of 5% Advance-DS, 5% Shinchrom E71, 5% or 10% Thermon 3000, the other was a glass capillary column (Shinwa Kako Co.) coated with films of DEGS (BCL type) or Thermon 3000 (WCOT type). Packed type column chromatography could not separate the fatty acid compositions of C20:0, C20:1, C20:3 and C20:4, whereas the glass capillary type column chromatography could clearly identify the individual compositions in the fatty acid. Especially, by means of the glass capillary column with Thermon 3000, the Δ8, 11, 14 C20:3 was separated from Δ11, 14, 17 C20:3. This indicates a possible usage of the latter composition as the internal standard in the gas liquid chromatography. Therefore, the glass capillary column chromatography seems to be more appropriate to qualitative and quantitative analysis of fatty acid compositions.
The above technique was applied to analyze blood samples of a healthy young man who was leaded with daily ingestion of canned mackerel meat rich in eicosapentaenoic acid (EPA) for 8 consecutive days. During the ingestion of mackerel meat, cholesterol, phospholipid, triglyceride and β-lipoprotein in the plasma showed a progessive decrease, while plasma HDL-cholesterol remained unchanged. Plasma arachidonic acid did not exhibit any significant change during. and after the meat ingestion. Platelet arachidonic acid, on the other hand, tended to increase for the second half of the ingestion period with its maximum value on the 2nd postingestion day. Plasma EPA reached its peak concentration on the 4th ingestion day.
A sharp increase in platelet EPA was observed during the ingestion period. Collagen induced platelet aggregation was reduced with the ingestion of mackerel meat. However, ADP induced platelet aggregation was not affected by mackerel meat.
These results lead us to the conclusion that EPA rich fish meat seems to play some roles in reducing the plasma lipid levels and in preventing the platelet aggregation.

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The determination of PGs concentration by RIA is often encountered by the problem of cross-reacting PG homologues, and of the facile interconversion among these homologues. We have made a slight modification of Jaffe's method utilizing a pretreatment technique consisted of the extraction by organic solvent and column chromatography on silicic acid.
The specimen was defatted wtih n-hexane, followed by PGs extraction into a mixture of ethyl acetate -isopropanol- water (7:3:6, v/v/v). The extract was then applied onto the minicolumn packed with silicic acid. The eluent employed was a mixed solvent of benzene, ethyl acetate, and methanol in variable ratios. The PGs were eluted in the order of PG (A+B), PGE, and PGF, separately.
The assays of PGs were performed by Clinical Assays' RIA kits, with the sensitivity of 20 pg/ml. Although the results of dilution test for urinary PGs have exhibited an excellent linearity, the serum PGs concentration showed falsely low values when the large specimen volume (over 1 ml) was employed. It seems to be due to the effect of nonspecific interference caused by the coexistent neutral lipids.
Using this method, we examined the serum PGE concentrations in congenital heart diseases and significantly high PGE levels were observed in some cases of tetralogy of Fallot, mitral insufficiency, and so forth.

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In the present study a selective determination of Thromboxane B₂ (TXB₂) and 6-Keto-Prostag, landin F_1α (6KF) in plasma was performed using the improved resolving power of highperformance thin layer chromatography (HPTLC) and sensitive radioimmunoassay.
TXB₂ and 6KF were extracted with ethyl acetate pH3.0, applied on a HPTLC plate and developped with ethyl acetate/acetic acid. The Rf values of TXB₂ and 6KF were 0.37 and 0.14 respectively and they could be separated from prostaglandin A₂ (PGA₂), PGB₂, PGE₂ and PGF_2α. The recoveries at each step of extraction by ethyl acetate and HPTLC fractionation were about 95% and 80%, and total recovery was about 72%. Normal ranges of TXB₂ and 6KF by this method were 154.6±34.5 (SD, n=24)pg/ml and 123.3±46.9 (SD, n=15)pg/ml respectively.
Plasma TXB₂ concentration in cerebral ischemia measured by this method rose appreciably in relation with its attack.
The features of the present method are the following: 1) The purification procedure by ion exchange column chromatography prior to HPTLC can be eliminated. 2) Simultaneous separation of TXB₂, 6KF, other prostaglandins and contaminants can be achieved on a HPTLC plate by ethyl acetate/acetic acid. 3) The simplified procedure brought a considerable reduction in time.

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The analysis of prostaglandins (PGs) were investigated by high performance liquid chromatography (HPLC) using 9-anthryldiazomethane (ADAM).
ADAM, which is the labeling reagent of carboxylic group, can react with PGs rapidly. The derivatives were separated in reversed phase chromatography using Zorbax ODS column (4.6mm i. d.×25cm). Methanol-water was found to be suitable for complete separation of ADAM esters of PGE₁, PGE₂, PGF_1α and PGF_2α within 30 minutes.
The PGs-ADAM esters can be detected by fluorophotometer at Ex. 365nm Em. 412nm.
In this method, the fluorescence intensity of the derivatives have wide linearity over 100ng and detection limit was 100pg.

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4-Bromomethyl-7-acetoxycoumarin (Br-Mac) reacted with prostaglandins (PGs) to form the ester derivatives in iacetone solvent in the presence of KHCO₃ and dibenzo-18-crown-6. The esters were separated by high-performance liquid chromatography (HPLC). Each lebeled cornpund eluted from a column was successively mixed with 0.1 N NaOH and hydrolyzed to the fluorescent coumarin derivative, and then introduced to a flow-through fluorometer (Ex 365, Em 460nm). In this system, whatever the kind of PG is, the fluorophore to be detected is common to all PGs. Therefore, the fluorescence quontum yield was hardly influenced by the PG moiety, compared with 4-bromomethyl-7-methoxycoumarin (Br-Mmc), which was previously reported as a fluorescence labeling reagent for HPLC of PGs. Gradient elution technique could be more effectively used in this system than Br-Mac method because the fluorescence intensity of this hydrolysate was not subject to the solvent effect. It was found that Br-Mac could react with all PGs and related compounds investigated in this work: PG F_1α, F_2α, E₁, E₂, D₁, D₂, B₁, B₂, A₁, A₂, 6-keto-F_1α, TX B₂ and arachidonic acid etc. Calibration graph of PGs showed good linearity at least from 1 nmol to 5 pmol and the detection limit was about 10 fmol. Attempts were made to apply this method to the determination of PGs in human seminal fluid. After deproteinization with methanol containing 16-methyl-PG F1α (internal standard), the supernatant was acidified with 0.1 N HCl (pH 3-4) and extracted with ethyl acetate. The organic phase was evaporated and then allowed to react with Br-Mac. An aliquot of the resulting solution was injected to a HPLC column. The peaks corresponding to PG F2α, E2 and E1 were found on the chromatogram, but any prominent peaks corresponding to PG B and A series (and their 19-OH analogs) could not be found, the occurence of which was previously reported. These results seem to agree with the suggestion of Jonsson et al. that some, if not all, of the PGs of the B and A series (and their 19-OH analogs) in human seminal fluid were artifacts.

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Radioimmunoassay (RIA) is a sensitive, specific and handy method, which has been widely utilized in the determination of prostaglandin (PG) and thromboxane (TX). In consideration of the disadvantage of RIA depending on the use of radioisotope we made attempts to apply enzyme immunoassay (EIA) to the assay of PG and TX, in which enzyme-labeled hapten molecule is used instead of radioactive compound.
The mixed anhydride reaction was performed to conjugate the amino group of β-galactosidase to the carboxyl group of PGF_2α, TXB₂ and 6-keto-PGF_1α, respectively. The enzyme-labeled antigen could be stored at 4° without an appreciable loss of enzyme activity and antigenicity for more than a year.
After competitive immunoreaction with antibody between enzyme-labeled antigen and unlabeled antigen the immunoprecipitate formed was separated by the double antibody method. The enzyme activity of the precipitate was assayed fluorometrically with 4-methylumbelliferyl-β-Dgalactoside as substrate. PGF_2α, TXB₂ and 6-keto PGF_1α could be measured quantitatively in the range of 0.06-30pmol, 0.1-30pmol, 0.14-30pmol, respectively. The sensitivity was comparable to that of RIA. The cross-reactivities with other PGs and their metabolites were less than 1% in most cases.
A column of octadecyl silica was used to extract TXB₂ which was added to human plasma, and the extract was subjected to RIA and EIA. A good correlation was obtained between the amount of added TXB₂ and that of measured TXB₂ (y=1.09x+11.07pmol/ml, r=0.99) A satisfactory interrelation was also observed between RIA and EIA (y=0.92x+4.64pmol/ml, r=0.96).

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The arachidonic acid (AA) metabolism of human platelets was studied under various experimental conditions by use of the thiobarbituric acid (TBA) reaction as well as a radioisotope technique, and a simple method was developed for estimation of exogenous and endogenous AA metabolism including platelet cyclo-oxygenase (PCO) pathway, platelet lipoxygenase (PLO) pathway and AA liberation from membrane phospholipids.
When AA was incubated with washed platelets cyclo-oxygenase of which had been completely inhibited by aspirin (ASA), TBA-reactive substance (TBARS) was not detected within short incubation periods, especially in alkaline media, although control (intact) platelets produced promptly a significant amount of TBARS even under such experimental conditions. Therefore, it was suggested that TBARS produced by the incubation of AA with control platelets for short incubation periods before the development of ASA-resistant TBARS in alkaline media was malondialdehyde (MDA) via the PCO pathway. When AA was incubated with the cyclo-oxygenaseinhibited platelets, prolongation of the incubation time especially in acid media produced a significant amount of TBARS, suggesting that TBARS thus obtained was produced via the PLO pathway.
These concepts were supported by the following experimental results: (1) When ¹⁴C-AA was incubated with platelets, time courses for the formation of PCO products were faster than those for the formation of PLO products and optimal pH values for the formation of PLO products were around 6 while those for PCO products were around 7.4;(2) Time course and pH profile for the production of TBARS by the incubation of AA with the ASA-treated platelets were similar to those obtained by the incubation of AA with the soluble fraction of platelet homogenates (PLO enzyme preparation);(3) Phenidone, a PLO enzyme inhibitor, inhibited the production of TBARS in both of these experiments, indicating that TBARS thus obtained was synthesized from AA by the PLO enzyme. When thrombin was incubated with washed platelets, TBARS was promtly produced and optimal pH values for its production were around 8. The TBARS formation by thrombin was completely inhibited by ASA, indicating that the thrombininduced TBARS (Th-TBARS) was MDA via the PCO pathway alone derived from endogenous AA liberatied by thrombin from platelet phospholipids. Therefore, Th-TBARS can be used as an indicator for estimating AA liberated from phospholipids when the PCO pathway is normal.
Based on these experimental results, the PLO and PCO pathways were estimated by TBARS determined after the incubation of 0.2mM AA at pH 6.5 for 10 min with 10⁸ platelets which had been incubated with 1mM ASA for 5 min at 37° and after the incubation of 0.2mM AA with 10⁸ untreated platelets at pH 7.4 for 1 min, respectively. AA liberation in cases of normal PCO pathway was estimated by TBARS determined after the incubation of thrombin (10U/ml) with 2×10⁸ platelets at pH 7.4 for 10 min. Normal values expressed in terms of nmol MDA were 1.17±0.34 per 108 platelets (m±SD, n=31), 0.79±0.15 per 108 platelets (n=31) and 0.64±0.24 per 2×108 platelets (n=23) for the PLO pathway, PCO pathway and AA liberation, respectively. The validity of the experimental conditions for estimation of the PLO and PCO pathways was confirmed by the results that PLO and PCO activities as determined by those conditions were not detected when platelets were obtained from PLO defective patients and from ASA-ingested normal subjects, respectively, and that TBARS production by normal platelets as an indicator of PLO and PCO pathways was strongly inhibited in vitro by phenidone and ASA, respectively.

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It has been reported that ω-3 polyunsaturated fatty acids, especially ω-3 eicosapentaenoic acid (ω-3, EPA), is a potent antagonist of platelet aggregation and therefore might reduce thrombotic disorders. In order to clarify the effect of ω-3 polyunsaturated fatty acids or EPA on the metabolism of prostaglandins and thromboxanes in platelets and vessel wanes, the present study was performed.
Highly purified (75%) EPA ethyl ester prepared from sardine fish oil was given to male wister rats (60mg/kg/day for 8 weeks). Plasma EPA concentration and EPA/AA ratio were higher in EPAEE fed rats than in control rats. Platelet aggregability in the rats fed EPAEE was significantly reduced. The generation activity of PGI₂ like substance from thoracic aorta of EPA fedrats was markedly higher than that from control rats. The conversion from ¹⁴C-arachidonic acid (AA) to ¹⁴C-6keto PGF_1α was higher in EPA fed rats, but not significant. There was no detectableconversion of ¹⁴C-EPA to ¹⁴C-17δ-6keto PGF_1α, both in EPA fed rat and in control rats. PGI₂ like substance generated from aorta of EPA fed rats was thought to be PGI₂ itself.
EPA rich fish oil concentrate prepared form sardine fish oil was given to 8 healthy male volunteers (1.4g EPA/day, for 4 weeks). EPA concentration both in plasma and platelet were significantly increased after 4 weeks ingestion of fish oil concentrate. Platelet aggregation induced by collagen and threshold dose of ADP were also significantly decreased. Conversion of both ¹⁴C-AA to ¹⁴C-TXB₂ and ¹⁴C-EPA to ¹⁴C-TXB₃ by washed human platelet were not changed during the experiment. The value of the conversion of ¹⁴C-EPA to TXB₃ was far lower than that of ¹⁴C-AA to ¹⁴C-TX132T. here was no detectable formation of ¹⁴C-TXB₃ from ¹⁴C-EPAlabeled platelet under the stimulation of collagen, while ¹⁴C-EPA was released from the platelet. TXB₂ formation from ¹⁴C-AA labeled platelet stimulated by collagen was significantly decreased after 4 weeks ingestion of fish oil concentrate. However the conversion of AA released from ¹⁴C-AA labeled platelet to TXB₂ was not changed during the experiment. On the other hand, the release of AA from ¹⁴C-AA labeled platelet was significantly reduced after the experiment.
These results indicate that the anti-thrombotic action of EPA and related compounds might be partialy explained by the enhancement of PGI2 production from vessel wall and the reduction of TXA₂ formation from platelet due to the reduced generation of AA from platelet membrane.

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Plasma thromboxane B₂ (TXB₂) in ischemic cerebrovascular diseases and other diseases were measured and its relation to the period after onset (or last attack) and to aspirin (ASP) treatment was studied. Platelet aggregation was examined simultaneously in some cases.
Object: For TXB₂, transient ischemic attack (TIA) 8 cases (11 measured), cerebral infarction 18 cases (19 measured), cerebral bleeding 2 cases, epilepsy 2 cases and normal healthy control 24 cases were measured. For platelet aggregation, 4 cases of TIA, 8 cases of cerebral infarction and other 3 cases were examined.
Assay methods: TXB₂ was extracted from heparinized plasma by ethyl acetate, purified by thin layer chromatography and assayed by radioimmunoassay. Platelet aggregation was examined from sodium citrate-mixed blood by absorption method for ADP (2×10^-5M) and collagen (3μg/ ml)
Result: Plasma TXB₂ (mean±SD) was as follows, control: 155±35pg/ml; TIA: ASP nontreated: 260±215 (6 measurement), ASP treated: 52±41 (4); cerebral infarction: within one month after onset: ASP non-treated: 1750±1310 (4), ASP treated: 42 (2), over one month after onset: ASP non-treated: 59±35 (7), ASP treated: 64±16 (3); cerebral bleeding: 6 days after onset: 1120, 9 months after onset: 165, epilepsy: one day after attack: 626, one month after attack: 298.
In eschemic cerebrovascular diseases (TIA and cerebral infarction) logarithmically converted values of both TXB₂ and the period (days) after onset were correlated signifcantly (correlation coefficient -0.77, N=15, p<0.01). Seven paired TXB₂ values of ASP non-treated and treated cases, measured at the same time after onset, were selected. Their means and SD were 236±205 in ASP non-treated and 51±23 in ASP treated cases. It's difference was not significant. 2 cases over 90% absorption in platelet aggregation for both ADP and collagen (TIA and epilepsy) gave high TXB₂ values (3320 and 298 respectively) and other 13 cases under 90% had normal values.
Conclusion: Plasma TXB₂ increased in ASP non-treated cases of TIA and those of cerebral infarction within one month after onset. Each case after cerebral bleeding and convulsion gave also high plasma TXB₂ values. Increased platelet aggregation was found in 2 cases showing high plasma TXB₂ values.

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We studied the pharmacological and biochemical effects of OKY-1581 which was a specific inhibitor of thromboxane synthetase. The following results were obtained.
IC₅₀ value for rabbit platelet thromboxane synthetase was 3X10-9 M. OKY-1581 inhibited the rabbit platelet aggregation induced by arachidonic acid (IC₅₀=5.8X10-7M). Rabbit sudden death induced by arachidonic acid (4mg/kg, iv) was protected by oral administration (30mg/kg). Inhibition of arachidonic acid induced vascular contraction was also observed by intravenous administration (0.01mg/kg). Ex vivo experiment using monkeys showed that OKY-1581 admini-stered orally at a dose of 0.5mg/Icg inhibited the TXB2 production in serum over 7 hours.
From these results, OKY-1581 could be useful for diseases such as angina, myocardial infarction and cerebral infarction in which the thromboxane synthesis was enhanced.

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The salidiuretic furosemide increases renal blood flow (RBF) and inhibits platelet aggregation. The increase in RBF is inhibited by indomethacin and thus may be attributable to the modification of renal prostaglandin (PG) metabolism by furosemide. The suppression of platelet function is also ascribed to the influence of furosemide on platelet PG metabolism. Piretanide is a new salidiuretic similar to furosemide in its pharmacological profile. In the present study, we examined the effect of piretanide on platelet function with special referenci to effects on platelet PG metabolism.
At the concentration of 5 x10-4M, piretanide inhibited platelet aggregation and ATP secretion induced by ADP, collagen, epinephrine and PGH2. The primary aggregation induced by ADP, however, was sustained in the presence of piretanide. The inhibitory effect of piretanide on ATP secretion was more prominent than that on platelet aggregation. For arachidonic acid-induced aggregation, complete inhibition was observed after the addition of piretanide if a low concentration (0.6mM) of arachidonic acid was employed. However, complete aggregation was transiently noted followed by disaggregation if a high concentration (3.6mM) of arachidonic acid was employed. ¹⁴C-serotonin release from platelets after the addition of ADP or collagen was almost completely abolished by 5x10^-4M piretanide. Piretanide at the concentration of 5x10^-4M also suppressed partially malondialdehyde formation induced by A23187 or thrombin. When platelets were incubated with ¹⁴ C-arachidonic acid in the presence of piretanide, the production of thromboxane B₂ measured by thin-layer chromatography was decreased whereas the production of PGF_2α and PGE₂ was increased. Analysis of thromboxane B₂ and PGD₂ by gas chromatographymass spectrometry also demonstrated the decreased production of thromboxane B₂ and the increased production of PGD₂. The slight increase in cAMP levels of platelets was observed after the addition of arachidonic acid. Piretanide enhanced this increase remarkably, but aspirin cancelled the enhancing effect of piretanide. Piretanide did not influence either adenylate cyclase activity or cAMP phosphodiesterase activity of platelets.
These results indicate that piretanide causes a partial inhibition of platelet thromboxane synthetase followed by reorientation of cyclic endoperoxide metabolism, that is, the decrease in thromboxane A₂ production and the increase in PGD₂ production which stimulates platelet adenylate cyclase. The inhibitory effect of piretanide on platelet function would result not only from reduced formation of thromboxane A₂ but also from increased production of PGD₂.

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Cultured porcine thyroid cells produce PGE₂, PGF_2α , TXA₂ and PGI₂. PGE₂ and PGI₂ stimulate c AMP synthesis and iodine metabolism. When cultured in the presence of TSH, the cells produce PGI₂ and when cultured in the absence of TSH, the cells produce PGE₂, PGF_2α , and TXA₂. TSH acutely stimulates PGE₂ and PGI₂ syntheses. When cultured in the presence of TSH, TSH acutely stimulates PGI₂ synthesis and when cultured in the absence of TSH, TSH acutely stimulates PGE₂ synthesis. In the presence of TSH, PGI₂ plays more important role than other PGs.

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The synthetase and dehydrogenase of prostaglandin (PG)D₂ has been demonstrated in brain, spinal cord and alimentary tract, contrasting with those of PGE₂ which has been previously investigated as a controller of endocrine pancreas. We examined the effect of PGD₂ on pancreatic islet function in perfused rat pancreas.
Only glucagon was strongly released by 14μ M of PGD₂ in the presence of 2.8mM glucose while both glucagon and insulin were released by 14μ M of PGE₂. When 5.6mM glucose was used, result were same except the release of glucagon was partly diminished. The dose of PGD₂ reduced to 1.4μ M, and glucagon release during 10 minutes was about one sixth of 14μ M PGD₂ in the presence of 2.8mM glucose.
In conclusion, PGD₂ released glucagon from perfused rat pancreas without insulin release, while PGE₂ did release both of insulin and glucagon. Considering the fact that suppression of endogenous PG by aspirin or indomethacin restores glucose tolerance in diabetics, PGD₂ but PGE₂ may be a controller of endocine pancreas.

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Prostaglandins (PGs) are widely distributed in biological organs, and plays an important role as cellular mediating factor.
In endocrinological organs they have been reported to be involved in hormone secretion. As for insulin secretion, however, the consistent effect of PGs on somatostatin release have not been reported to our knowledge.
Therefore, 20 islets isolated from Wistar male rats, 250-300g in body weight, were perifused to evaluate a role of PGs in pancreatic hormone release.
The standard perifusate was Krebs-Henseleit bicarbonate buffer, pH7.4, containing 0.25% bovine serum albumin and 8.3mM glucose.
Perifusion speed was 0.5ml/min. and the perifusate was collected in a test tube every 2 minutes.
In the experiment, the islets was perifused with PGE₁ and PGE₂ of 10^-7M to 10^-5M added in the standard medium for 14 minutes each.
PGE₁ and PGE₂ at the present concentration did not change insulin secretion pattern of the control.
Sum of insulin secretion for 14 minutes was not affected by these PGs also.
Somatostatin secretion was not influenced by the PGE₁ and E₂ as well.
In order to evaluate a role of endoginous PGs in insulin secretion sodium salicylate, which inhibits PG synthesis, was added in the standard perifusate.
Insulin secretion was by 50-60% decreased by this treatment.
These supression was recovered to the level prior to the administration of sodium salicylate by the addition of 10^-6M PGE₁ and PGE₂.
Therefore, the endogenous PGE₁ and PGE₂ might be involved in insulin secretion, although a role of endogenous PGs in somatostatin release was not clarified by the present investigations.

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Glycosylation of human hemoglobin has been calimed, as well as it was reported that the level of glycosylated hemoglobin was elevated in the individuals in poor diabetic control.
Structual studies in human hemoglobins have shown that hemoglobin is a tetrameric protein consisting of two alpha and two beta subunits, which exists in two allosteric forms, one called T and the other did R form. In the physiological condition hemoglobin in the deoxy state is the T form with which heme-iron exists in high spin, on the other hand in the oxy one is the R form with which heme-iron in low spin. Between these two forms there are differences in physical properties so that those are able to identify from each other by a certain technique such as visible or ultraviolet spectroscopy.
Many organic phosphates are known as effective allosteric effector site binding substances, also many of which, for a example, inositol hexaphosphate (IHP) binds so tightly to the binding site that the conformation of the tetrameric protein of hemoglobin causes a shift from the R to the T allosteric form. This reaction may be failed in glycosylated hemoglobins, since these substances (i. e. IHP) binding to glycosylated hemoglobin can be blocked by the glucose residue covalently attached to the N-terminus of the beta chains. The relative amount of glycosylated hemoglobins present in the blood sample can be determined from the absorption spectrums taken before and after addition of a prefered allosteric effector site binding substance.
We evaluated the A-gent from ABBOTT KK, the kit for to determine the concentration of glycosylated hemoglobins consists imidazole buffer with potassium ferricyanide and a detergent, IHP solution and three calibrators. The potassium ferricyanide oxidizes the hemoglobin, and a detergent lyses the red blood cells to release homoglobins. The imidazole coordinates with the heme-iron shifting equilibrium allosteric isomers to the R form. Addition of IHP which reacts with the allosteric binding site of major (non-glycosylated) hemoglobin, causes a shift of the equilibrium of allosteric isomers to the T form.
The absorption spectrums at 560nm and 633nm are measured before and after addition of IHP to the treated sample. The concentration of glycosylated hemoglobin in the specimens may be given from the standard curve of three calibrators.
For this study we utilized ABBOTT-VP automated analyzer and the specimens from both diabetic and non-diabetic subjects. Three specimens were tested from different classes to carry out the precision study, their mean values were 632, 7.92 and 12.13% glycosylated hemoglobin and±S. D.(or±C. V.) values were respectively 0.39 (6.10%), 0.32 (3.98%) and 1.03 (8.51%). The regression statics for 40 clinical specimens between the values obtained by the proposed method and those by HPLC method gave the correlation coefficient, 0.659 and the linear regression appeared its slope, 1.7 and Y-intercept, 6.7.
This method is able to be automated and may has the advantages in simple and rapidly performance of the glycosylated hemoglobin test.

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For the determination of glycosylated hemoglobin, we studied a spectrophotometric “Automated-procedure” by using phytic acid. Then, the above procedure was compared with an isoelectric focusing method, a mini-column method and an electrophoretic method.
As anti-coagulants in this procedure, EDTA-2Na and EDTA-2K were used for the determination of hemoglobin A₁ concentration but not heparin sodium salt, sodium fluoride, sodium citrate nor potassium oxalate. Variations of intra-assay were good (CV=1.96-2.68%). Variations of inter assay were suitable (CV=5.34-6.34%). The correlation between phytic acid method and the other three methods were shown on the following lines (a, b, c),
(a) isoelectric focusing method (r=0.934, y=1.07X+1.32),
(b) mini-column method (r=0.918, y=1.00X+0.29),
(c) electrophoretic method (r=0.970, y=0.87X-0.41).
Comparison of glycosylated hemoglobin values in normal subjects and diabetics were reviewed as follows;
(a) isolelectric focusing method: normal subjects: 5.04±0.75%HbA₁C (mean±SD), n=34 diabetics: 8.81±2.37%HbA₁C, n=53
(b) mini-column method: normal subjects: 6.54±0.93%HbA₁, n=89 diabetics: 10.28±2.52%HbA₁, n=160
(c) electrophoretic method: normal subjects: 7.33±0.80%HbA₁, n=37 diabetics: 12.24±3.49%HbA₁, n=36
(d) phytic acid method: normal subjects: 6.37±1.03%HbA₁, n=74 diabetics: 10.76±2.58%HbA₁, n=160 (“n” indicates the number of subjects)
The concentrations of glycosylated hemoglobin (HbA₁) by phytic acid method were compared in well controlled, moderately controlled and poorly controlled diabetics. The significant differences (p<0.001) were observed between the poorly controlled (12.26±2.09%) and moderately controlled diabetics (9.69±1.90%) or poorly controlled and well controlled diabetics (9.02±1.65%).
The above findings suggest that this “phytic acid method” is suitable for the routine use of the determination of hemoglobin A₁ values in clinical laboratories.

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A new chromatographic method for the determination of glycosylated hemoglobin was developed in our laboratory. In this method, IEX-530CM (Tôyôsôda Co.) was used instead of Bio Rex 70 cation exchange resin (Bio Rad Inc.) and gradient elution was performed with 30mM sodium phosphate, pH6.50 buffer and 30mM sodium phosphate containing 0.2M NaCl, pH6.50 buffer. Blood samples were obtained by venipuncture and hemolysates were prepared by the method of Goldstein13). Hemoglobins of the blood sample were divided into about sixteen fractions within twentyeight minutes by our new method. Characteristic of each hemoglobin fraction was examined before and after the pretreatment of normal and diabetic erythrocytes in vivo and in vitro. Four major labile glycosylated hemoglobins were found in blood red cell, namely, L-GHb4, L-GHb7, L-GHb9 and L-GHb12. And stable glycosylated hemoglobin (St-GHb8) was separated from the labile glycosylated hemoglobin (L-GHb7), which were commented as two chromatographically indistinguishable forms by Goldstein et al. The Hb6 fraction was identified as HbF by its properties against alkaline conditions and by its chromatographical kinetics. More than one percent of HbF occured in the samples from normal and diabetic subjects. The Hb10 fraction was found to increase fairly high after the incubation of erythrocytes with saline at 37° for 6hrs. Comparing the fraction of hemoglobin divided by McDonald et al.21) with our fractions, A₁a₁ corresponded to Hb₁, A₁a₂ to Hb2, A₁b to Hb3, L-GH4 and Hb5, A₁c to HbF, L-GHb7, St-GHb8, L-GHb9, Hb10 and Hb11, A₀ to Hb12-Hb16, respectively. Fluctuations in A₁c and A₁ were due to variable amounts of components, which we know here were L-GHb4, HbF, L-GHb7 and L-GHb9. Recently, removal of labile fractions by dialysis of hemolysate or by saline inculation of red cells was recommended. But with these procedure Hb10 increases fairly high, as mentioned above, and is not separated from A₁ or A₁c by the conventional methods.
For the correct interpretation of glycosylated hemoglobins, it is necessary to mind these factors which flucturate and complicate the observed values or to measure the stable glycosylated hemoglobin (St-GHb8) directly. Otherwise the obtained results will not be accurate indicators of longterm blood glucose control.

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In diabetics, the concentration of HbA₁C gives valuable information on the regulation of carbohydrate metabolism during a period of 8 to 12 weeks before its determination, and it has recently come to be recognized as a useful parameter in the management of patients with diabetes mellitus.
Of the various methods developed for estimating HbA1C, the one most frequently cited is that of Trivelli and her co-workers. It is, however, a time-consuming method, and in addition, very complicated technology. High-performance liquid chromatography, that is, HPLC, has also been used in the assay of glycosylated hemoglobin. This technique is not as time-consuming, and is more precise. Furthermore, it requires only a minute amount of blood. However, the ion exchange resin usually used in this sytem, Bio Rex 70, is too soft to bear repeated loads of high pressure. Gel deformation may occur, resulting in the clogging of the column and failure to sustain a constant flow. Inaccurate data is produced after the column has been used many times.
Recently, we synthesized a new type of hydrophobic stationary phase to be used for this kind of HPLC assay. This stationary phase, Micropearl SFWA₁C, is composed of crosslinked synthetic organic polymers and the separation mechanism is based upon mixed interaction which are reversed phase partition chromatography and ion-exchange chromatography. The polymers are hard enough to withstand repeated used and are able to make a better separation of minor component of hemoglobin in a shorter elution time compared to the convetional method. The HgA₁C values obtained by this column are correlated well with those of obtained by Bio Rex 70 column.
Employing this Micropearl SFWA₁C column, the HbA₁C automated analyzer has developed. It is operated by a microcomputer control which includes an alarm system with self-checking of pressure, flow volume, and data processing. It has a snake-chain type auto-sampler which carries cups of the samples. The system employs bichromatic measurements (one at 415nm and one at 500nm), using the difference in the two measurements to eliminate error introduced by variations in the light source. In this system, only 3μl of whole blood is required as the specimen. The specimen is added to 450μl of the hemolytic reagent, which is an aqueous solution of sodium azide. The resultant sample can be injected directly into the injection valve.
As for the precision data of this method, the coefficient of variation of the intraassays for normal subjects was 3.23%, and 3.38% for diabetic subjects. The coefficient of variation of the interassays for normal subjects was 3.85%, and 3.90% for the diabetics. Next, from the histogram of HbA₁C in normal Japanese subjects, we calculated the normal range of this hemoglobin component as the mean, 4.83±0.94%, which is twice the standard deviation. The correlation between the values for present HbA₁C levels and fasting blood sugar levels measured at various times, the best correlation was found for the blood sugar level measured two months before the sample for the HbA₁C assay was taken. Good correlation was found for values from one month before, and fair correlation for three months before. HbA1C values correlated least well when compared with values for FBS measured on the same day.
As mentioned above, this method has the merits of a simple procedure and a column which can be used repeatedly, and should prove itself useful in a wide variety of clinical situations.

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Determination method of the HbA₁ by electrophoresis (1) using the Agar-Gel Film developed by Coming was investigated.
1) Reagents and Apparatuses Buffer Solution: 0.1M Citrate buffer, pH 6.2 Support Medium: HbA₁ Agar-Gel Film (Corning) Hemolyzing Agent: 0.1% Saponin and 0.05% EDTA Electrophoresis Chamber: U-shaped electrophoresis chamber Densitometer: M-720 Densitometer (Corning)
2) Measuring Method The hemolysate is obtained by adding 300 μl of saponin to 100μl of blood collected in EDTA. 1μl of hemolysate thus prepared is applied to the Agar-Gel film and is electrified at 60V for 40 minutes. The film is then dessicated in the oven and is put on the densitometer at 420nm for obtaining the HbA₁ value.
3) Results
The within-day precision of this method was x=7.26%, C. V. =2.20% in healthy subjects and =10.71%, C. V.=1.69% in diabetic patients.(Table 1) The between-day precision was x=7.09% x, C. V. =2.76% in healthy subjects and x=9.44%, C. V. 1.79% in diabetic patients.(Fig. 1)
The blood sample collected in EDTA was immediately hemolyzed. The hemolysate thus prepared was kept at 4°. No fluctuation was observed on HbA₁ value for 14 days.
No significant difference was noted among the HbA₁ value at 4°, 10°, 20° and 37° of buffer solution.(Fig. 2)
It is known that the disposable columns (mini-column) allow bilirubin to influence on the measurement value, whereas no influence was noted up to its amount of 30mg/dl in this method.
Comparative studies were made on the blood sample for HbA₁ measurement. The average values in healthy subjects were x=6.76% with disposable columns (2)(mini-column) and Y=6.93% with this method. The correlation coefficient between the two methods was γ=0.96 with the regression equation, Y=0.90X+0.89. The average value in diabetic patients were x=9.75% with disposable columns and Y=10.05% with this method. The correlation coefficient between the two methods was γ=0.98 with the regression eqaution, Y=1.10X-0.68.(Fig. 3)
The average values in healthy subjects were x=6.69% with the column method (3) and Y=6.94% with this method. The correlation coefficient between the two methods was γ=0.96 with the regression equation, Y=0.98X+0.41. The average value in diabetic patient was x=9.60% with column method and Y=10.05% with this method. The correlation coefficient between the two methods was γ=0.97 with the regression equation, Y=1.12X+0.66.(Fig. 4)
The average HbA₁ value was x=4.70% with the column method (3) and the average HbA₁ value was Y=6.94% with this method both in healthy subject. The correlation coefficient between the two months was γ=0.96 with the regression equation, Y=1.03X+2.11. The average HbA₁C value was x=7.49% with the column method and the average HbA₁ value as Y=10.05% with this method in diabetic patient. The correlation coefficient between the two methods was γ=0.96 with the regression equation, Y=1.12X+1.66.(Fig. 5)
4) Conclusion
From the aforementioned experimental results, it can be said that the Agar-Gel Electrophoresis, which requires so less amount of sample as 100μl and gives the favorable reproductibility, is simple, time-saving, satisfactorily endurable to the influences of the temperature and bilirubin, and highly correlated with the disposable column method or column method. Therefore, it can conclusively be assessed that this method is an attractive and useful alternative to the other methods for the routine measurement of HbA₁.

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Three aspects were under consideration through daily experiences.
Regarding to unstable HbA₁ in microcolumn chromatography, further improvements in accuracy and specificity should be rather required, otherwise at least in present method, there were no significances in removing unstable HbA₁ by pretreatment of erythrocytes.
In the cases of HbA₁ elevation aside from diabetes mellitus, determinations of HbA₁ subcomponents were desirable and subsidiary index of HbA_1a+b/HbA_1c ratio was available.
From long-term follow-up study, it was demonstrated that HbA_1c has proved to be more useful than FBS in assessing the degree of patient control, especially under insulin therapy.

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Nonezymatic glucosylation of plasma protein was determined with tiobarbituric acid (Fluckiger and Winterhalter). Plasma glucose should be removed out for avoiding cross-reaction with the thiobarbituric acid. Glucose-free plasma was hydrolyzed after 4.5 hour-boiling and deproteinzed with 40% trichloracetic acid. Supernatant was incubated with 0.05 M thiobarbituric acid at 40° for 40 min. The reaction product was absorbed at 443nm with a spectrophotometer. Hydroxymethylfurfural (HMF) was used as a standard and glucosylated plasma protein was expressed as nmol HMF/mg protein. Plasma protein was measured with the method of protein dye binding (Bradford). Glucosylated hemoglobin (HbA₁) was determined with agar gel electrophoresis (Lionel).
In 83 diabetic patients newly diagnosed glucosylated plasma protein was 1.58±0.5nmol HMF/mg protein which was significantly higher than that in healthy subjects or impaired glucose tolerance (IGT). Hb A₁ in diabetic patients was 10.86±2.4%. There is significant correlation between Hb A₁ and fasting blood glucose or Hb A₁ and 2 hour-blood glucose during 75 g GTT.
In 96 diabetic patients having been treated with sulphonylurea or insulin the average value of blood glucose measured frequently at the past three months was significantly correlated with Hb A1 (r=0.77, P<0.001).
Glucosylated plasma protein also was significantly correlated with the average value of blood glucose measured at the past three months in the diabetic patients (r=0.45, P<0.05). Because a turnover of plasma protein is shorter than the life span of blood red cells, the past three monthaverage value of blood glucose is correlated more tightly with Hb A₁ than glucosylated plasma protein.
Blood red cells of healthy subjects were washed by physiological saline, and glucose solution was added to them at each concentration of glucose, 100, 300 and 500mg/100ml. The mixture was incubated at 37° and glucosylation of hemoglobin began 24 hours after the incubation with glucose as well as glucosylation of protein.
In 13 diabetic patients blood glucose, Hb A₁ and glucosylated plasma protein were determined every week for 4 weeks after the beginning of treatment. At 4 weeks the blood glucose decreased by 48% but Hb A₁ and glucosylated plasma protein slightly decreased by 18% and 29%, respectively. Improvement of Hb A₁ is very slow as compared with blood glucose.
The measurement of Hb A₁ is very useful for evaluation of diabetic control. However, in anemia and abnormal hemoglobinemia such as thalassemia, Hb A₁ value is erroneous. On the other hand glucosylated plasma protein reflects on the condition of diabetes at the past 1 or 2 weeks. When the glucosylated plasma protein is measured with thiobarbituric acid, because TBA reacts to glucose, it is necessary to exclude glucose from plasma with dialysis.
Based on our findings we suggest that Hb A₁ or glucosylated plasma protein should be measured monthly at least. Glucosylated plasma protein may be an adequate index of diabetic control as well as Hb A₁. Any increase in glucosylated plasma protein may indicate impairment of diabetic control. Further study is necessary to prove the worth of detecting early diabetic microangiopathy by measurement of glucosylated protein.

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In normal subjects and patients with diabetes mellitus (type II), HbA_1a+b, HbA_1c and HbA₀ were fractionated by column chromatographic procedure of Trivelli et al and HbA₁ was determined by a commercial kit. Moreover, the total glycosylated hemoglobin (GHbA), glycosylated hemoglobin in HbA₀ (GHbA₀), glycosylated serum protein (GSP) and urinary glycosylated protein (UGP) were determined the colorimetric method of Gabbay et al, using thiobarbituric acid.
Although there was no significant relation between HbA_1a+b levels and blood sugar levels, HbA_1c levels correlated significantly with the average blood sugar levels for several months proceeding the hemoglobin measurements. A significant correlation existed between HbA_1a+b and HbA_1c (r=+0.54, p<0.01), and, therefore, a striking correlation was evident in the plots of HbA_1a+b+HbA_1c against the mean blood sugar levels for 3 months prior to the hemoglobin measurements, which had a correlation coefficient of 0.79 (p<0.001). In addition, HbA₁ levels correlated significantly with GHbA (r=+0.89) and with GHbA₀(r=+0.94). On the other hand, the correlation coefficient between HbA₁ and GSP was only +0.41, although it was statistically significant (p<0.01). In particular, HbA₁ levels were not parallel to GSP levels in diabetics complicated with liver diseases. These results suggest that HbA₁ (HbA_1a+b+c) determinations by a commercial kit are a useful indicator for assessing the long-term blood glucose control in diabetics.
In non-diabetics with nephrosis or chronic nephritis, the ratio of UGP to SGP averaged 0.95±0.17. The marked elevation of the ratio (1.95±0.53) was evident in diabetics with nephropathy, showing the elevated UGP levels in these patients. The finding suggest that the glycosylated protein might play a role in pathogenesis of diabetic nephropathy.

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Not only patients with diabetes mellitus but also patients with renal failure had high hemoglobin A₁ (HbA₁) levels.
The high performance liquid chromatographic quantification of components of the HbA₁ showed that the fractional pattern of HbA₁ in patients with renal failure was similar to that seen in normal subjects rather than in diabetic patients.
In diabetic patients, there was a positive correlation between HbA₁ values obtained by the column method and those obtained by the thiobarbituric acid (TBA) method. However, although patients with renal failure had high HbA₁ levels, the patients showed HbA₁ levels similar to those observed in normal subjects, according to the TBA method.
Then, the formation of HbA₁ based on incubation of red blood cells with urea and other substances was observed. It was revealed that HbA₁ in patients with renal failure could be formed by Hb and cyanic acid originated from urea.
In addition, as non-enzymatic glycosylation may be a generalized reaction, we utilized the reaction in which glucose is linked to the amino group of Hb and obtained the result, that glucose could attach to the amino group of 3, 5, 3'-L-triiodothyronine (T₃), a thyroid hormone. Moreover, we also demonstrated that glycosylated T₃ could be separated from T₃ by high performance liquid chromatography.
The non-enzymatic binding reaction between various amino acids and glucose as observed in the present study seem to be a common reaction in the living body.

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Effects of renal failure on HbA_I levels were investigated.
Criteria for glucose intolerance with normal HbAI levels were determined: normal OGTT or borderline OGTT with FBS below 117mg/dl, peak BS below 223mg/dl and ΣBS below 980mg/dl. By these criteria 30 patients with non-hemodialysed, non-diabetic CRF were selected and their HbA_I levels were measured by ion-exchange column chromatography using Trivelli's method. HbA_I levels elevated in patients with BUN levels above 50mg/dl and correlated significantly with BUN (r=0.83, p<0.0001) and CRTN (r=0.57, p<0.001).
Red blood cells from normal subject were incubated for 5 days in 30mM triethanolamine HCl buffer, and HbA_I levels were determined. In medium containing 500mg/dl of urea N (containing tracer dosis of ¹⁴C-urea) and 100mg/dl of glucose, HbA_Ia+b, HbA_Ic and the leading edge of HbA_II increased markedly. HbA_Ia+b increased to 12.0% from 2.0%, and HbAIC to 19.2% from4.7%. ¹⁴C-urea was incorporated into HbA_Ia+b, HbA_Ic and the leading edge of HbA_II. The ratios of D. P. M./ml to optical density of each eluate, which might imply numbers of urea molecule per hemoglobin, were greater in HbA_Ia+b and HbA_Ic than in the leading edge of HbA_II. In media with urea N levels above 100mg/dl (100, 200, 300, 400 and 500) HbA_I levels correlated highly with urea concentrations (r=0.99, p<0.0001). Addition of other uremic toxins (CRTN, UA, MG, GAA, GSA and GPA) to the incubation media did not significantly increase HbA_I levels.
These findings suggest that urea induces the elevation of HbA_I levels due to the direct binding of urea to hemoglobin or due to the binding of cyanate, which is in equilibrium with urea in aqueous solution, and that HbA_Ia+b and HbA_Ic might bind more urea (or cyanate) molecules than the leading edge of HbA_II. In diabetic CRF patients, therefore, an incraese in HbA_I might be influenced not only by impaired glucose metabolism but also by high BUN levels.

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