Cholate rates

The reconstituted system consisted of Cytochrome P-488 (0.2 nmol), NADPH-Cytochrome c reductase (1500 units) and sodium cholate (1.25 mg). It was preincubated for 30 min at 31°. The final reaction mixture (which was incubated at 31° for 20 min) com tained the preincubated system described above, excess NADPH and t4C-BP (100 nmol 4.1 mCi/mmol) in a final volume of 1 mL 0.5M HEPES buffer, pH 7.6. Rate of BP metabolism was 665 pmol/min/nmol Cytochrome P-488. Abbreviations used for metabolites are described in legend to Figure 2. 

As expected, axial OH groups were easier to differentiate from equatorial ones than equatorial OH groups from one another. In the case of methyl cholate 66a, a standard reagent (pyridine, 50) does a good job. But in the glucose derivative 67a, the two equatorial OH groups are much more similar to one another. Therefore it is not surprising that they react with almost the same rate in the uncatalyzed reaction. When pyridine (50) was used as catalyst, the acylation of the 2-position (67c) was preferred by a factor of 4 but also a bis-acylated product 67d was formed. Concave pyridine 3r showed the best results. With a selectivity of 9 1, the 2-acylated product 67c was formed and no diacylated product 67d could be determined. 

Mixture B K[SiPh(3-fcat)2 and K[SiPh(dbcat)2] (3-fcat 2,3-dihydroxybenzaldehyde, dbcat 3,5-di-f-butylcatechol) contained two complexes with asymmetric catechols. Each complex showed the presence of two resonances due to the isomerism described above. The equilibrated mixtures showed the presence of two further species (Figure 9). These are attributed to isomers of the [SiPh(3-fcat) (dbcat)]- anion. Equilibrium was not established even after 8 weeks, whereupon decomposition prevented a more quantitative kinetic analysis. Flowever, it is apparent from the two experiments described that the kinetics of redistribution of ligands between complexes varies dramatically according to the cate-cholate involved. It is reasonable to conclude that the rate of redistribution decreases as the strength of the catecholate derivative increases. The nonstatistical distribution of complexes in a mixture indicates a thermodynamic stability of the complexes in Me2SO. The likely explanation lies in the electronic rather than the steric effects in the complex, since the live-coordination imposes little steric constraint. 

S. acidocaldarius (strain 7) contains a cyanide-sensitive cytochrome oxidase [24], The purified cytochrome (M, 150000) is composed of three subunits (M, 37000, 23 000, and 14000). Difference spectra following reduction with dithionite show a Soret band at 441 nm and a maximum at 603 nm characteristic of aa3-type cytochromes. In addition, there is a band at 558 nm whose connection to the oxidase is not clear. This oxidase is stimulated by cholate, but unlike the oxidase from the DSM 639 strain it is inhibited by low concentrations of cyanide (pM as opposed to mM) and oxidizes horse-heart cytochrome c, TMPD-ascorbate, and caldariella quinol. The rates of oxidation (pmol/min/mg protein) for cytochrome c, TMPD-ascorbate, and quinol are 63, 6.1, and 0.2, respectively. Another cytochrome oxidase that has an absorption maximum at 602 nm, oxidizes caldariella quinol, but does not oxidize cytochrome c, is also present in strain 7 so that the terminal portion of the electron transport system in S. acidocaldarius consists of at least three oxidases. It is suggested [8] that the presence of three oxidases in 5. acidocaldarius is unlikely and that the cyanide-sensitive oxidase was isolated from a different species, namely S. solfataricus. There is little taxonomic information in this assertion to judge whether strain 7 and DSM 639 are indeed different species. However, based on growth conditions reported by the investigators [12,28], which are unique for S. acidocaldarius and S. solfataricus [ 22, there is no reason to suspect that these organisms are different species. 

Gel permeation. Bio-Gel A-5m (57 x 2.5 cm column) was equilibrated in the appropriate buffer and packed in a column. Three ml sample of membranes was applied to the top of the column and the column irrigated with 40 Tris-HCl-40 mM NaCl-0.24% deoxy-cholate (DOC) at a flow rate of 10 drops min". Fractions containing 100 drops (approximately 1.7 ml) were collected. 

The rate of cholate synthesis in man is about 200-300 mg/day, as measured by isotope dilution studies. The chenodeoxycholate synthesis rate is similar, so that the total primary bile salt synthesis is about 400-600 mg daily for a healthy adult, and when in the steady state this amount is also the daily fecal excretion rate (12). 

Values for turnover time, pool size, and half-life of cholate and chenodeoxycholate and the excretion rates for both are given in Table I. When turnover time or synthesis rates are calculated from isotope dilution studies, the values are usually in the same range as those derived from isotope balance studies provided that the latter are done by properly validated and reproducible methods. 

Patients with hypercholesterolemia do not appear to have significant alterations in bile salt synthesis rates, but patients with combined hypercholesterolemia and hypertriglyceridemia have increased synthesis rates for both cholate and chenodeoxycholate (20). Bile salt synthesis rates are not appreciably changed when nicotinic acid feeding lowers plasma cholesterol concentrations (20). Synthesis rates may also be affected by thyroid hormones. Cholic acid synthesis is decreased and half-life prolonged in hypothyroid subjects. These alterations may be corrected with thyroid hormone (21). Bile acid synthesis is increased in thyrotoxicosis (21). 

As will be discussed in a later section, patients with certain types of liver injury have chenodeoxycholate as the predominant primary bile salt in their serum. Primary bile salt concentration ratios in serum are a fairly accurate reflection of the primary bile salt concentration ratio in bile. The evidence for this is given in Fig. 3, in which the primary bile salt concentration ratio in serum is plotted against that in bile in 14 patients. The correlation coefficient for these two variables is 0.86 (p<0.01). When chenodeoxycholate predominates in bile its metabolites (lithocholate and others) predominate in feces, and when cholate predominates in bile its metabolites (de-oxycholate and others) predominate in feces (27). This relationship is shown in Fig. 4. It thus appears that primary bile salt concentrations in blood and bile are related to their relative synthesis rates and that the predominant bile salt in blood and bile has the greater synthesis rate, since its metabolites predominate in feces. This assumes of course that there is a steady state and... 

Urinary excretion of bile salts by healthy subjects is apparently very limited. The urine contained 2 % of the radioactivity administered orally as i C-cholic acid to a healthy subject in whom 100% of the radioactivity was recovered (80) and 0.12% of radioactivity administered to four normal subjects when i C-cholate was given intravenously (25). Conventional methods do not detect bile salts in the urine of healthy subjects (81,82). In jaundice patients, however, bile salts are excreted in the urine regularly (83). The highest 24-hr excretion rates reported by Gregg occurred in patients with common bile duct obstruction (58 mg/24 hr) and drug-induced cholestasis (40 mg/24 hr). The cholate/chenodeoxycholate ratio was greater than 0.59... 

Fig. 5. Rate of disappearance of sodium tauro-cholate-24-i C from the enterohepatic circulation 24 and 48 hr after intravenous injection into a patient with active regional enteritis without resection (E. C.)...

Fig. 6. Rate of disappearance of sodium taurocholate-24-from the enterohepatic circulation 24, 48, and 72 hr after intravenous injection into a patient with Whipple s disease before and 3 months after treatment with antibiotics (left panel). Composition of residual radioactivity in duodenal fluid obtained at 3, 24, 48, and 72 hr after the intravenous injection of sodium taurocholate-24-i C into patient E. C. with Whipple s disease before and 3 months after antibiotic treatment (right panel). Data are expressed in terms of percentage of radioactivity contributed by each bile salt fraction to the total recoverable i C radioactivity. GDC, glycodeoxycholate GC, glyco-cholate TDC, taurodeoxycholate TC, taurocholate. In part from Garbutt et al. (9).

Since, in the rat, cholesterol is eliminated largely in the form of bile acids, it was expected that bile acid secretion in bile would be increased in the hyperthyroid state. Early experiments to test this point indicated that biliary bile acid secretion was actually normal or below normal (2,3). These results can be explained in terms of the inadequate analytical procedures then in use. Only cholate secretion was measured, and the levels of cheno-deoxycholate were not taken into account. When both of these bile acids were determined, it was shown that, in the bile fistula rat, the total production of bile acids was about the same in the hyperthyroid as in the euthyroid state, and lower in the hypothyroid state (4). In addition, in the hyperthyroid state, the normal ratio of cholate/chenodeoxycholate was reversed from approximately 3 1 to 1 3—cholic acid synthesis was decreased, and chenodeoxycholic acid synthesis was increased two- to threefold (4). Identical results were obtained in the bile fistula rat treated with noncalorigenic doses of D-tri-iodothyronine (5,6), suggesting that these effects are not necessarily a function of the basal metabolic rate. 

By injecting normal and hypophysectomized rats with i C-labeled cholate and/or chenodeoxycholate, the half-life, turnover rates, and bile acid pool sizes were determined (23-25). In hypophysectomized rats, bile acid synthesis (calculated from the /1/2 and turnover rates) and excretion are reduced to about half when compared to normals. The decrease in bile acid synthesis is reflected in drastically reduced levels of chenodeoxycholate Apparently, the hypophysectomized rat loses its ability to synthesize this bile acid (23,24). Typical values for daily bile acid synthesis expressed in mg/day/100 g rat were 0.55 mg cholate, 0.19 mg chenodeoxycholate for normals, and 0.31 mg cholate, no detectable chenodeoxycholate for hypophysectomized rats. 

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