sensitive and simple high-performance liquid chromatographic determination method for D-penicillamine in serum was developed. The method is based on the fluorescence derivazation of D-penicillamine with N-[4-(6-dimethylamino-2-benzofuranyl) phenyl] -maleimide and following fluorescence detection by high-performance liquid chromatography. A calibration graph for D-penicillamine was linear over the range from 200 fmol to 12 pmol, and the lower limit of detection at a signal-to-noise ratio of 2 was 50 fmol. Recovery rate of 2.9μM of D-penicillamine was 91.8% (C.V.=4.2%, n=5). Time courses of serum levels of D-penicillamine after single oral administration of D-penicilamine in capsule to three women were examined. Serum levels reached maximum after 2 hr and then decreased gradually till 24 hr. This method will be useful for a monitoring of D-penicillamine in blood.

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17-Ketosteroid-Sulfates (17-KS-S) and 17-Hydroxycorticosteroid-Glucuronides (17-OHCS) vaues in 24 hrs and morning urines were measured in normal adults (21-75 yrs. old) with maximal blood pressure between 110 and 139 mmHg and degree of obesity of 0.86-1.15 and in subjects with hypertension, hypotension, obesity. hyperlipemia and diabetes mellitus.
1) In normal adults the 17-OHCS/creatinine value showed only little changes according to age of subjects and the 17-KS-S/creatinine value decreased with increasing age of subjects and both values showed no significant difference when regarding sex. The 17-KS-S/17-OHCS ratio decreased with increasing age of subjects while the sex difference was insignificant too.
2) In subjects with hypertension, hypotension, obesity and diabetes mellitus the 17-KSS/17-OHCS was, mainly due to low values of 17-KS-S/creatinine, significantly lower than that in age-matched controls and especially showed clearly low values in hyperlipemic subjects, hypertensive subjects with obesity and subjects in advanced cancer of stomach, etc.
These lead to the conclusion that 17-KS-S/17-OHCS ratio must be considered as a prime parameter of adrenocortical function, because shifts of the patterns of adaptation of the human body in aging, the above diseases etc. are reflected in changes of the ratio.

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A newly developed enzymatic method for the determination of total bile acids in serum using 3α-Hydroxysteroid dehydrogenase (3α-HSD) and 3-oxo-5β-steroid Δ⁴-dehydrogenase (Δ⁴-DH) is described. This method is based ona principle that nitrotetrazolium blue (NTB) is reduced to diformazan with Δ⁴-DH, catalyzing Δ⁴-dehydrogenation of 3-oxo-bile acids. The sensitivity of this method is twofold higher than that of the method using a system of diaphorase-NTB with NAD-dependent 3α-HSD. The proposed method shows a satisfactory recovery (100.1%), a good reproducibility (CV of 4.55% for 11μmole/l) and a good correlation (r=0.995, n=137) to the previous method.

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Interrelationship of serum concentrations between carotenoids and lipid peroxides (LPO) was investigated in 36 healthy adults (19 males and 17 females, aged 33-72 years). Serum concentration of α-carotene or β-carotene measured in June was significantly correlated with that in December 1987 [α-carotene: r=0.830, p<0.001; β-carotene: r=0.953, p<0.001]. Biannual serum concentration of lycopene or LPO was also significantly correlated, but the correlation coefficients were relatively smaller [lycopene: r=0.418, p<0.05; LPO: r=0.472, p<0.01]. Serum β-carotene level was inversely correlated with serum level of retinol binding protein (RBP) [r=-0.381, p<0.05]. Negative correlation was also observed between serum LPO level and serum β-carotene [r=-0.347, p<0.05] or α-carotene [r=-0.437, p<0.01]. Serum LPO was positively correlated with serum RBP [r=0.462, p<0.01]. These finding may suggest that serum α-and β-carotene, but not retinol, might reduce lipid peroxidation. Such a mechanism could possibly be involved in the well-known negative association between dietary β-carotene and cancer risk.

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Activities of Asparate aminotransferase (AST), Alanine aminotransferase (ALT) and Lactate dehydrogenase (LDH) and isoenzymes of AST and LDH were determined in the liver homogenates of ICR mice. Vitamin B-6 group was also examined in the homogenates by HPLC, dividing into pyridoxamine (PM), pyridoxal (PL) and pyridoxine (PN). Activities of AST and ALT increased in the liver with age, while LDH showed no change. In LDH-isoenzymes, LD-5 decreased, and hybrid-isoenzymes increased with age. A significant correlationship was observed between AST and ALT in the liver. Although there was no significant correlation between s-AST and ALT, a marked relationship was significantly observed between s-AST and ALT. Interestingly, blood glucose levels correlated with hepatic ALT activi-ties, but not the AST activities.
In the liver PL accounted for 70% of the B-6 content, and PM was 30%. B-6, PM, and PL highly correlated to each other in the liver. The ratio of apo-ALT increased well above that of AST in the liver.

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We have previously reported a patient with anatomically-proved myocardial infarction in which no elevation of serum creatine kinase (CK) activity was demonstrated. Isoenzyme study revealed an absence of both CK-MB and CK-MM activity in the serum.
In this paper we determined immunoreactive CK-MM by the enzyme immunoassay in serum as well as tissues of caridiac, skeletal and rectal muscles of this patient to investigate whether the deficiency of MM isoenzyme activity is due to inhibition of activity or to deficiency of MM isoenzyme protein.
The amount of the CK-MM protein was markedly decreased in both serum and muscles, while enolase ft-subunit and S100ao, the specific marker antigens for the cardiac and skeletal muscles, were normally present. The complete absence of CK-MM protein in the serum and muscles were also confirmed by SDS-PAGE protein stain stain as well as immunoblotting.
Deficiency of the CK-MM protein appeared not to be fatal as seen in this patient. Compensation for the deficiency of CK-MM could be carried by CK-BB, mitochondrial CK or an alternate pathway of ATP production through adenylate kinase in the muscle tissues.

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We extracted α-ketoaldehyde dehydrogenase (α-KAD) from rabbit's liver (New Zealand White, body weight 3kg). The α-KAD reacted with methylglyoxal as a substrate in the presence of coenzyme NAD with a Km value of 0.24 mM. Preincubation of glycated bovine serum albumin as a substrate with NAD and α-KAD at 0°C or 30°C for 2 hours caused significantly smaller increases in fluorescence intensity after subsequent incubation for 7 days at 37°C, compared with samples preincubated without α-KAD at 37°C. Preincubation of glycated bovine serum albumin for 2 hours with α-KAD and NAD at 30°C caused the smallest increase in fluorescence intensity after 7 days of subsequent incubation.
These findings suggest that α-KAD may inhibit the formation of the advanced Maillard products.

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A rapid micromethod for apo E phenotyping directly from plasma has been established. Ten μl plasma were delipidated, dried and then solubilized in 10 mM Tris-HCl buffer with 8 M urea. Isoelectric focusing (IEF) in 5% polyacrylamide flat gel with 6.4M urea and 2.8% pharmalyte (PH 4-6.5, Pharmacia) was carried out, as reported previously. After IEF, the flat gel was washed in a blot buffer (0.7% acetate), followed by Western blotting on nitrocellulose membrane in the same blot buffer using the Trans-Blot Cell (Bio Rad Co.). Then, immunostaining was performed using goat-anti-human apo E (Daiichi Chemicals) as first antibody and biotinylated anti-goat lgG as second antibody, VECTASTAIN ABC Kit (VECTOR Laboratories), and 4-chloro-1-naphthol as a substrate. As a result, six apo E phenotypes were clearly demonstrated. It took only 2 days for apo E phenotyping. Thus, this method is suitable for routine examination and large scale screening for studying the relationship between apo E phenotypes and hyperlipoproteinemia and atherosclerosis

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