We report a new micro-scale (0.1-mL sample) turbidimetric method for determination of protein by use of benzethonium chloride in alkali. The method is highly specific for protein, has a higher sensitivity than the classic method of Lowry et al., and shows satisfactory reproducibility and recovery. The turbidity produced in our method is the same for albumin and gamma-globulin and is more stable than in Meulemans' method (in which sulfosalicylic acid is used) or in the method of Bossak et al. (in which trichloracetic acid is used). In contrast to Pesce and Strande's method, there is no manipulative loss of protein.
A previously unknown HPLC peak was recently observed in urine samples from patients with Cushing's syndrome and disease. We analysed dansylated derivatives of 17keto steroid glucuronides in urine samples from patients with Cushing's syndrome, Cushing disease and from healthy subjects using high-performance liquid chromatography (HPLC) on reversed-phase Cap Cell PakC8. All urine samples from patients with Cushing's syndrome caused by adrenal adenoma and Cushing's disease showed an unknown large peak at the point between [110HE-G] and [110HA-G] peaks and at a retention time of 25.4 min. The same unknown peak was also observed in urine samples from a patient with asymptomatic cortisol-producing adrenal adenoma and two patients with ectopic ACTH-producing tumor, though the peak height was low for the former and one of the latter but high for the second of the two patients. In contrast, healthy male and female urine only showed a very small peak at the same retention time. Urine samples from a Cushing disease treated with op'DDD and Cushing's syndrome bilaterally adrenalectomized and treating with cortisol showed no such peak. The retention time of this unknown peak is clearly different from that of seven 17keto steroid standard glucuronide conjugates. The structure of this substance may be closely related to [110HE-G] or [110HA-G].
Abstract. We developed a new method for measuring an unidentified ketosteroid glucuronide (US-G) detected by the method of Iwata et al. for measuring 17-ketosteroid glucuronides by reversed phase HPLC on a Capcell-Pak C8 column with three kinds of mobile phase solutions (Iwata method; Clin Chem 35: 795-799,1989). The Iwata method inadequately separated US-G and two hydroxy 17-ketosteroides, 11/3-hydroxyetiocholanolone and 11/3-hydroxyandrosterone, and it exhibits insufficient sensitivity for measuring traces of US-G in the urine of healthy subjects. We solved these problems by developing a new method which measures US-G in urine, as a free type by hydrolyzing the glucuronide type enzymatically, by normal phase HPLC on a Capcell-Pak Silica column with one kind of mobile phase solution. By this method, the levels of US excreted as a glucuronide in the urine of healthy subjects and of patients with Cushing's syndrome were determined as proportions of the levels of 11/3-hydroxyandrosterone.The average daily urinary excretion of US was 971 jig (125-4,995 jig) in patients with Cushing's syndrome (n=22: two males and 20 females aged 26 to 65 years), and 34 jig (0-141 jig) in healthy subjects (n=63: 49 males, and 14 females aged 21 to 54 years), and the differences were clearly significant. However, there were no differences between the urinary US levels of patients with pituitary adenoma and patients with adrenal adenoma. Furthermore, no US was detected in the urine of patients with aldosteronism (two males and eight females aged 34 to 61 years). The daily level of urinary US excretion in two of the patients with polycystic ovary syndrome was 159 and 142 µg, but no US was detected in the other two patients.
We previously reported that the daily urinary unidentified ketosteroid glucuronide (US-G) level in patients with Cushing's syndrome was much higher than that in the healthy subjects. Furthermore, urine samples from patients with Cushing's syndrome, including those with pituitary adenoma and adrenal adenoma, yielded almost the same high excretion levels, despite the different sites of the adenomas. We extracted US obtained by hydrolysis of US-G in urine of patients with Cushing's syndrome, purified it, and analyzed its chemical structure. Molecular weight and molecular formula were analyzed by MS spectrometry, and the chemical structure was analyzed by NMR spectrometry, utilizing small quantities of refined US. The substance has a molecular weight of 304 Da, a molecular formula of C19H28O3, and its chemical structure is 3alpha,11beta-dihydroxyandrost-4-en-17-one.
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