William C. Duane

Effect of regulating cholesterol biosynthesis on breath isoprene excretion in men

Isoprene is a normal constituent of human breath and may be derived from the cholesterol synthetic pathway. Acute and chronic lovastatin and a cholesterol-supplemented diet were used to determine whether a mechanistic link exists between isoprene and cholesterol biosynthesisin vivo in humans. The acute effects of lovastatin, a competitive inhibitor of the rate-limiting step of cholesterol biosynthesis, on breath isoprene excretion was determined by administering a single 20, 40 or 80 mg dose of this drug to five healthy male subjects at 8 p.m. and measuring their breath isoprene levels every 4 h for one 24 h cycle before and after treatment. When compared to the baseline cycle, all three doses of lovastatin significantly reduced breath isoprene levels at 6 and 10 h post-drug treatment. Chronic lovastatin therapy (40 mg b.i.d. for 6 wk) reduced 6 a.m. breath isoprene levels (time of maximum baseline value) by 27 ± 9% (SEM) and cholesterol synthesis measured in freshly isolated mononuclear leukocytes (ML) by 12 ± 6%. A cholesterol-supplemented diet (1070 mg, total) ingested for 6 wk reduced breath isoprene excretion and ML sterol synthesis by 16 ± 5 and 19 ± 4%, respectively. The parallel decreases in isoprene excretion and cholesterol synthesis caused by these pharmacologic and dietary means suggest that breath isoprene is derived from the cholesterol synthesis pathway.

Distribution, turnover, and mechanism of renal excretion of amylase in the baboon

Pure amylase was isolated from pancreata and parotid glands of the baboon, an animal which has a serum amylase level and renal clearance of amylase (C(Am)) similar to man. After bolus injection, both pancreatic and salivary amylase rapidly disappeared from the serum in a monoexponential fashion with a mean serum half-time of approximately 83 min. Only about 24% of the amylase cleared from the serum appeared in the urine indicating that the majority of amylase was removed from the serum by an extraurinary mechanism. The C(Am) by the kidney was constant over a wide range of serum amylase levels and the ratio of C(Am)/C(In), which averaged 3.0%, was not influenced by mannitol diuresis. This suggests that the renal excretion of amylase results from glomerular filtration without appreciable tubular reabsorption. Pancreatic amylase was consistently cleared more rapidly by the kidney than was the baboons endogenous amylase while salivary amylase was consistently cleared less rapidly than endogenous amylase. THE FINDINGS IN THIS STUDY PROVIDE INSIGHT INTO SEVERAL OF THE FOLLOWING CLINICALLY OBSERVED PHENOMENA: (a) the short serum half-time of amylase accounts for the transient nature of serum amylase elevations in pancreatitis; (b) the extra-urinary removal of amylase accounts for the maintenance of relatively normal amylase levels in uremia; and (c) the more rapid renal clearance of pancreatic amylase compared to salivary amylase may explain the disproportionate elevation of the urinary amylase excretion rate relative to the serum amylase level in acute pancreatitis.

The intermicellar bile salt concentration in equilibrium with the mixed-micelles of human bile

The intermicellar bile salt concentration in equilibrium with the bile salt-lecithin-cholesterol mixed-micelle has been studied in human bile. Equilibrium-dialysis, used to measure the biliary intermicellar bile salt concentration, has been validated as an applicable method by studying the cholate-lecithin mixed-micelle, for which intermicellar bile salt concentration values have previously been reported. The intermicellar bile salt concentration of bile was essentially independent of ionic strength in the range 0.05-0.15 M chloride. Simple dilution of bile lowered the intermicellar bile salt concentration (about 2/3 reduction for each two-fold dilution). This reduction occurred because of a simultaneous decrease in the molar ration of bile salt/phospholipid in the micelle. Dilution of micelles with micellar bile salt/phospholipid held constant did not affect the intermicellar bile salt concentration. The relationship between intermicellar bile salt concentration and micellar bile salt/phospholipid, defined in the dilution studies, was linear in the range of study. For a composite of five biles, this relationship was described by the equation: intermicellar bile salt concentration = 1.27 (bile salt/phsopholipid) + 0.538. Data obtained on an artificial bile agreed closely with the results obtained on bile suggesting that the other constituents of bile did not affect this analysis. These findings may be helpful in understanding the process of micellar cholesterol solubilization in bile.

Effects of lovastatin and dietary cholesterol on sterol homeostasis in healthy human subjects.

We measured biliary and fecal sterol outputs in 12 human subjects on a metabolic ward in four randomly allocated, 6-7 wk periods: (a) lovastatin (40 mg b.i.d.) + low cholesterol diet (mean 246 mg/d), (b) lovastatin + high cholesterol diet (mean 1,071 mg/d), (c) low cholesterol diet alone, (d) high cholesterol diet alone. In addition to lowering serum LDL cholesterol, lovastatin significantly lowered biliary secretion of cholesterol, fecal output of endogenous neutral sterols, cholesterol balance, and systemic cholesterol input (the sum of cholesterol synthesis and absorbed dietary cholesterol). The high cholesterol diet significantly lowered cholesterol balance, but significantly increased systemic cholesterol input and fecal output of acidic sterols. There was no significant interaction between lovastatin and dietary cholesterol for any parameter measured. Judging from these data, the primary action of lovastatin is to lower cholesterol synthesis and systemic cholesterol input, the main compensatory response being reduced biliary cholesterol secretion. Conversely, increased dietary cholesterol appears to increase systemic cholesterol input, the major compensatory response being increased bile acid synthesis. There appears to be no interaction between these two perturbations of systemic cholesterol input.

Lovastatin added to ursodeoxycholic acid further reduces biliary cholesterol saturation

The effects of lovastatin and ursodeoxycholic acid on cholesterol saturation of gallbladder bile were examined, alone and in combination. Nine volunteers were studied before any treatment and after each of three treatment periods: lovastatin, 40 mg, twice a day; ursodeoxycholic acid, 10 mg/kg per day; and the combination of both drugs. Treatment periods were randomly ordered, lasted 4-5 wk, and each was preceded by a 3-wk washout period. Mean cholesterol saturation index decreased from a baseline value of 1.40-0.92 on lovastatin (p less than 0.008). Mean cholesterol saturation index on ursodeoxycholic acid was 0.87 and decreased to 0.70 with the addition of lovastatin (p less than 0.030). There was a strong correlation (r = 0.87, p less than 0.003) between saturation index on ursodeoxycholic acid and the further incremental reduction in saturation index with addition of lovastatin. These findings raise the possibility that addition of lovastatin to ursodeoxycholic acid treatment might improve the efficacy of this bile salt for dissolution of cholesterol gallstones, especially in patients with a suboptimal response to ursodeoxycholic acid.

Simultaneous study of the metabolic turnover and renal excretion of salivary amylase- 125 I and pancreatic amylase- 131 I in the baboon.

The metabolic turnover of salivary and pancreatic amylase was studied in the baboon, an animal with a serum amylase level and renal clearance of amylase similar to man. Purified amylase was electrolytically iodinated. Although iodinated and uniodinated amylase had similar gel filtration, electrophoretic, enzymatic, glycogen precipitation characteristics, the labeled enzyme was cleared less rapidly by the kidney than was the unlabeled material. However, urinary iodinated amylase which had been biologically screened by the kidney had a renal clearance and serum disappearance rate indistinguishable from unlabeled amylase and thus can serve as a tracer in metabolic turnover studies. Administration of a mixture of salivary amylase-(125)I and pancreatic amylase-(131)I made it possible to simultaneously measure the serum disappearance and renal clearance of these two isoenzymes. The metabolic clearance of both isoenzymes was extremely rapid with half-times of about 130 min. This rapid turnover of serum amylase probably accounts for the transient nature of serum amylase elevation which frequently occurs in pancreatitis. Pancreatic amylase-(131)I was consistently cleared more rapidly (mean clearance ratio: 1.8) by the kidney than was salivary amylase-(125)I. This more rapid renal clearance of pancreatic amylase may help to explain the disproportionate elevation of urinary amylase relative to serum amylase observed in pancreatitis.

Effects of soybean protein and very low dietary cholesterol on serum lipids, biliary lipids, and fecal sterols in humans

Soy-base texturized vegetable protein (TVP; Archer Daniels Midland, Decatur, IL) has been used to decrease serum cholesterol and as a substitute for animal protein to achieve very low levels of dietary cholesterol. The effect of very low dietary cholesterol and of TVP on biliary lipids and fecal sterols is unclear. The study objective was to determine the effects of very low intake of dietary cholesterol, as well as TVP itself, on serum lipids, biliary lipids, and fecal sterols. We studied eight normal subjects living on a metabolic ward during three randomly ordered 6- to 7-week periods: (1) standard cholesterol diet (190 to 550 mg/d), (2) TVP-low-cholesterol diet (17 to 30 mg/d), and (3) TVP-standard cholesterol diet. By analysis of covariance (ANCOVA), reducing dietary cholesterol to these very low levels significantly decreased serum low-density lipoprotein (LDL) cholesterol (P=.048) but did not affect high-density lipoprotein (HDL) cholesterol or triglyceride. TVP resulted in a borderline significant reduction in LDL cholesterol (P=.058) with a highly significant reduction in HDL cholesterol (P=.004) and an increase in serum triglyceride (P=.010). During TVP ingestion, there was a highly significant increase in the output of fecal neutral sterols (P=.005) and a tendency for a higher output of fecal acidic sterols (P=.100). Fecal sterol balance was significantly more negative (indicating increased cholesterol synthesis) during TVP ingestion (P=.016). Neither TVP nor the very-low-cholesterol diet appreciably affected the gallbladder bile molar percent cholesterol or saturation index. The data are consistent with the hypothesis that to the extent TVP decreases serum LDL cholesterol (an effect of borderline significance in this study), the effect occurs via a reduction in the absorption of cholesterol and perhaps bile acid. However, the potential benefit of decreasing LDL cholesterol in this way seems to be at least partially offset by a concomitant reduction in HDL cholesterol and an increase in serum triglycerides.

Floating Stools — Flatus versus Fat

Abstract Factors influencing the density of stools from 33 healthy subjects (nine had floating and 24 sinking stools) and six patients with steatorrhea were investigated. All floating stools sank when their gas volume was compressed by positive pressure. Thus, to float, stools must contain gas. After degassing, previously floating and sinking stools had similar specific gravities, indicating that the floating or sinking propensity of such stools depends upon differences in gas rather than fat content. A high stool gas content (and hence a floating stool) in healthy subjects appeared related to colonic methane production. Steatorrheic stools had a relatively normal gas content. The density of their nongaseous fraction was less than normal, but resulted primarily from increased water rather than fat content. Thus, stools float because of an increased content of gas or water (or both); the floating stool should not be considered a sign of steatorrhea.

Role of the unstirred layer in protecting the murine gastric mucosa from bile salt

Bile salts disrupt a functional gastric mucosal barrier which normally minimizes back-diffusion of H+ into mucosa. Our previous studies have shown that ionized bile salts disrupt the barrier to H+ by dissolving membrane lipids. The presence of an unstirred water layer on the surface of the gastric mucosa could protect against bile salt injury either by creating a concentration gradient of bile salt from lumen to mucosal surface or by slowing diffusion of lipid-laden mixed micelles away from the mucosal surface. In the present study we investigated this possibility in the anesthetized rat. Measurements of H+ back-diffusion and Na+ forward-diffusion across the gastric mucosa were made before and after exposure to a bile salt solution that was either unmixed or mixed by continuous withdrawal and injection. Using carbon monoxide diffusion, we observed this method of mixing to decrease the unstirred layer thickness from 880 to 448 micron (p less than 0.02). Mixing increased mean H+ back-diffusion induced by a 10 mM mixture of six conjugated bile salts from -2.58 to -4.11 microEq/min (p less than 0.01) and increased mean forward-diffusion of Na+ from 1.81 to 3.27 microEq/min (p less than 0.01). Mixing also increased efflux of mucosal phospholipid (32.7 to 52.2 nmol/min, p less than 0.05) and of cholesterol (4.89 to 8.87 nmol/min, p less than 0.05) into the bile salt solution. Addition of saturation amounts of lecithin and cholesterol to the bile salt solution completely prevented disruption of the barrier whether the solution was mixed or not. Mixing also increased mucosal uptake of bile salt from 74.6 to 221.3 nmol/min (p less than 0.01) when no lipids were added. In the presence of lecithin and cholesterol, mixing increased absorption of bile salt from 63.5 to 165.6 (p less than 0.02). These findings further support the hypothesis that bile salts disrupt the gastric mucosal barrier by dissolution of mucosal membrane lipids, and provide evidence that the unstirred water layer helps protect the gastric mucosa from bile salt injury.

Lack of benefit of ursodeoxycholic acid in drug-induced cholestasis in the rat.

Abstract The administration of ursodeoxycholic acid (UDCA) has been reported to improve cholestasis in patients with primary biliary cirrhosis or sclerosing cholangitis. In the present study, we tested the hypothesis that UDCA similarly might reduce cholestasis induced by drugs. Rats were treated with three different drugs reported to induce cholestasis: 17α-ethynylestradiol, α-napthylisothiocyanate, and cyclosporine A. UDCA administration (0.4·g/day-1 k-1 before and during administration of the cholestatic drug) did not improve survival, food intake, or serum indicators of cholestasis in any of these three animal models of cholestasis. To the extent that drug-induced cholestasis in rats mimics the human situation, we conclude that UDCA probably will not be beneficial in drug-induced cholestasis in humans.