Biochemistry Gallstones Case File

Biochemistry Gallstones Case File

Eugene C.Toy, MD, William E. Seifert, Jr., PHD, Henry W. Strobel, PHD, Konrad P. Harms, MD

❖ CASE 31

A 45-year-old female presents to the clinic with concerns over occasional midepigastric discomfort and nausea/vomiting after eating “greasy meals.” The symptoms gradually disappear, and she has no further discomfort. She denies any hematemesis, and pain is worse after eating. She further mentions that she had elevated cholesterol levels in the past and was on an exercise program, but not anymore. On exam, she is afebrile with normal vital signs. Her physical exam is completely normal with no evidence of abdominal pain. An abdominal ultrasound is performed and revealed a few gallstones in the gallbladder but no thickening of the gallbladder wall.

◆ What factors would you need to consider to assess the need for cholecystectomy?

◆ What are gallstones made of?

◆ Can gallstones be seen on abdominal x-ray?

ANSWERS TO CASE 31: GALLSTONES

Summary: A 45-year-old female presents with hypercholesterolinemia, ultrasound evidence of gallstones, and recurrent symptoms of gallbladder disease.

◆ Surgical candidates: Frequent and severe attacks, previous complications from gallstones, presence of underlying condition predisposing the patient to increased risk of gallbladder disease.

◆ Components of gallstones: Cholesterol, calcium bilirubinate, and bile salts.

◆ Abdominal x-rays and diagnosis: Mixed stones much easier to see on plain film secondary to calcifications, comprising approximately 10 percent of gallstones.

CLINICAL CORRELATION

This individual fits the “classic” patient with gallbladder disease: female, middle-aged, overweight. The gallbladder acts to store bile salts produced by the liver. The gallbladder is stimulated to contract when food enters the small intestine; the bile salts then travel through the bile duct to the ampulla of Vater into the duodenum. The bile salts act to emulsify fats, helping with the digestion of fat. Gallstones form when the solutes in the gallbladder precipitate. The two main types of stones are cholesterol stones and pigmented stones. Cholesterol stones are usually yellow-green in appearance and account for approximately 80 percent of gallstones. Pigmented stones are usually made of bilirubin and appear dark in color. Patients may have pain from the gallstones, usually after a fatty meal. The pain is typically epigastric or right upper quadrant and perhaps radiating to the right shoulder. If the gallbladder becomes inflamed or infected, cholecystitis can result. The stones can also travel through the bile duct and obstruct biliary flow leading to jaundice (yellow color or the skin), or irritate the pancreas and cause pancreatitis.

APPROACH TO BILE SALT METABOLISM

Objectives

1. Know about bile salt metabolism.

2. Be able to identify where bile salts are synthesized.

3. Know where bile salts emulsify dietary fats.

Definitions

Bile salts: Cholesterol derivatives with detergent-like properties used to solubilize cholesterol, assist in intestinal absorption of fat-soluble vitamins, and emulsify dietary lipids passing through the intestine to enable fat digestion and absorption by exposing fats to pancreatic lipases.

Bile acids: Neutral, protonated form of bile salts.

Primary bile acids: Synthesized from cholesterol as cholic acid and chenodeoxycholic acid. They are secreted as taurine and glycine conjugates.

Secondary bile acids: Products of deconjugated and reduced primary acids. Bacteria in the intestine remove the 7α-hydroxyl group, leaving the secondary bile acids.

Cholesterol 7α-hydroxylase (CYP7A1): The mixed-function oxidase cytochrome P450 enzyme catalyzing the initial, rate-limiting step for conversion of cholesterol to bile acids.

β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA) reductase: The rate-limiting enzyme in cholesterol biosynthesis.

DISCUSSION

Bile salts are derivatives of cholesterol that make up the major component of bile. They are very efficient detergents when conjugated to amino acids because of the presence of both polar and nonpolar regions. This property helps to emulsify dietary lipids in the intestine, which aids in fat digestion and absorption by making fat vulnerable to pancreatic lipases. Other functions of bile salt (ionized, deprotonated form) and bile acid (neutral, protonated form) are to solubilize cholesterol thus preventing precipitation of cholesterol crystals and facilitating cholesterol excretion. The only significant removal of excess cholesterol from the body is achieved through the excretion of bile salts. Finally, they assist in intestinal absorption of fat-soluble vitamins.

The conversion of cholesterol to bile acids is a multienzyme process. The initial and rate-limiting step of bile acid synthesis is oxidation of cholesterol to 7α-hydroxycholesterol by a mixed function oxidase from the cytochrome P450 superfamily, cholesterol 7α-hydroxylase (CYP7A1; Figure 31-1). The remaining steps include reduction of the Δ⁵-double bond, side chain shortening, and oxidation. The products of this pathway, cholic acid and chenodeoxycholic acid, are termed the primary bile acids because they have been synthesized de novo from cholesterol. To increase the pH range over which the bile salts remain ionized and serve as good detergents, they may be conjugated via amide bonds with either of the amino acids glycine or taurine (Figure 31-2).

Bile salts/acids are synthesized in the liver, stored and concentrated in the gallbladder, and secreted into the intestine where they may undergo deconjugation and reduction by intestinal bacteria to produce secondary bile acids (see Figure 31-1). Once formed in the liver, the bile salts/acids are

Figure 31-1. Simplified biosynthetic pathway of bile salts.

Figure 31-2. Conjugation of bile salts.

secreted through the bile ducts, which are made up of the bile canaliculi and bile ductules, and passed on to the gallbladder for storage as bile. Ultimately, they are passed on to the intestine. Most of the bile acids/salts are deconjugated in the intestinal ileum and reduced to secondary bile acids by bacteria, which remove the 7α-hydroxyl group by a dehydroxylation reaction. Although some are lost by excretion, approximately 90 percent of the bile acids and salts are reabsorbed in the terminal ileum and returned to the liver. The liver cannot provide sufficient amounts of newly synthesized bile acids for the body’s daily needs; therefore, the body relies on enterohepatic circulation (Figure 31-3) to sustain necessary bile acid levels. The portal vein transports bile acids from the intestine back to the liver as complexes with serum albumin.

The synthesis of bile salts is under very tight regulatory control to maintain cholesterol homeostasis and supply sufficient amounts of detergent to the intestine. This is controlled in part by a feedback mechanism on cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthetic pathway. Increased concentrations of bile acids inhibit cholesterol 7α-hydroxylase, while low levels relieve the inhibition. Elevated cholesterol levels, however, activate the enzyme, thus increasing bile acid biosynthesis. Levels of both cholesterol and bile acids affect the concentration of cholesterol 7α-hydroxylase and this regulation appears to be controlled at the transcriptional level via nuclear receptors. Therefore, binding of bile acids or cholesterol to a given nuclear receptor in turn regulates the expression of the CYP7A1 gene, activating expression in the case of binding to cholesterol and repressing it when bile acids are bound. Thus, the proper maintenance of bile acid levels can prevent accumulation of cholesterol.

Eighty percent of gallstones in the Western world are a result of cholesterol precipitation from the bile, a condition known as cholelithiasis. The pathogenetic mechanism of gallstone formation usually involves a culmination of

Figure 31-3. The enterohepatic circulation of bile acids.

deleterious events involving the metabolic pathways of cholesterol or the bile acids/salts. First, the cholesterol concentration in bile becomes supersaturated. Bile is a controlled mixture of cholesterol, bile acids, and phospholipids (with small amounts of bile pigments), and if cholesterol levels are elevated or bile acids/salts lowered, the ratio of the three major components changes leaving cholesterol less protected against the aqueous environment and more likely to precipitate. Elevated cholesterol levels can occur by having excess HMG-CoA reductase activity, the rate-limiting enzyme in cholesterol biosynthesis; this condition is typically seen in the obese. Alternatively, reduced levels of acyl- CoA: cholesterol acyltransferase [ACAT], the enzyme that esterifies cholesterol within cells, or reduced levels of cholesterol 7α-hydroxylase can cause elevation of cholesterol. Deoxycholate, a secondary bile acid synthesized by intestinal bacteria, inhibits CYP7A1. Therefore, high levels of deoxycholate  resulting from prolonged exposure of bile acids to intestinal bacteria may result in high levels of cholesterol in bile. In addition to elevated cholesterol levels in bile, there must be adequate time for cholesterol crystal nucleation, which will ultimately form macroliths. Fasting, like overnight sleeping, allows for long-term storage of bile in the gallbladder and could give ample time for crystal nucleation.

COMPREHENSION QUESTIONS

[31.1] The modification to bile salts that increases the working pH range and amphipathic nature of bile salts is

A. 7α-Hydroxylation

B. Dehydroxylation by intestinal bacteria

C. Esterification

D. Conjugation to taurine or glycine

[31.2] A new drug called CT2033, a corticosteroid, has reached the clinical trial stage. This drug was designed to treat inflammation but seems to also cause an undesired side effect where there is disturbance of cholesterol and bile acid homeostasis. Which of the following is least likely to explain the side effects caused by CT2033?

A. It downregulates the expression of a hepatobiliary bile acid transporter gene.

B. It inhibits a hepatobiliary transporter protein decreasing bile acid secretion.

C. It competes with cholesterol for CYP7A1 binding.

D. It binds and inhibits pancreatic lipases.

[31.3] A 53-year-old male patient with elevated levels of low-density lipoprotein (LDL) cholesterol, signs of premature cholesterol gallstone disease and substantially elevated triglycerides visited his physician for a follow-up to check his current status. The patient had received various statin, HMG CoA-reductase inhibitors therapies for the past 2 years. However, after blood work done at this follow-up visit, complications had still not subsided. This patient has similar problems as two of his siblings. Which of the following best explains this patients dyslipidemia? 

A. An influx of abnormal phospholipids in the gallbladder as a result of ileal disease

B. A loss of HMG-CoA reductase function

C. A loss of CYP7A1 (cholesterol 7α-hydroxylase) function

D. Elevated levels of ACAT

Answers

[31.1] D. Conjugation of bile acids to these two amino acids through amide linkages is important for maintaining the detergent properties of bile salts over the wide pH range of the intestinal tract. Conjugation decreases the pKa of the bile salts assuring ionization and solubility in the intestines.

[31.2] D. Any of the situations in answers A to C could directly alter cholesterol and bile acid homeostasis. Many of the bile acid transporters are regulated by nuclear receptors. Therefore, a nuclear receptor ligand such as CT2033 could change the expression levels of the transporters, which could then result in problems with secretion of bile acids and probable accumulation of cholesterol. If the new drug were to directly bind and inhibit the transporter, the same result would occur. Since corticosteroids are derivatives of cholesterol, it could be reasonable for CT2033 to fit in the same CYP7A1 binding pocket and compete with cholesterol. This could cause accumulation of cholesterol because CYP7A1 is needed to convert cholesterol into bile salts. The drug could act to effectively lower active CYP7A1 levels. However, answer D is least likely to affect cholesterol and bile acids since pancreatic lipases are involved in the breakdown of fat. Bile acids simply emulsify fats and allow pancreatic lipases to degrade the fat. Therefore, inhibiting pancreatic lipases should not have much of an effect on cholesterol and bile acid homeostasis.

[31.3] C. A loss in function of CYP7A1 prevents the catabolism of cholesterol to bile salts. Elevated levels of LDL cholesterol, signs of premature cholesterol gallstone disease, and substantially elevated triglycerides are all complications that can result from blocking the enzyme that breaks down cholesterol. Therefore, high levels of cholesterol accumulate in the bile and, with decreased production of bile salts to help dissolution of cholesterol, the formation of cholesterol gallstones. Statin therapy is not as effective because it inhibits the enzyme that controls the rate of cholesterol synthesis but does nothing with respect to the degradation of cholesterol. The increase in blood triglyceride levels when CYP7A1 is deficient is not well understood, but triglyceride levels appear to have a reciprocal relationship to bile acid synthesis. Finally, this appears to be a genetic disorder since other siblings showed the same phenotype, which would point to a possible mutated gene and likely prevent function of CYP7A1.

BIOCHEMISTRY PEARLS

❖ Bile salts are the major component of bile and are very efficient “detergents” when conjugated to amino acids because of the presence of both polar and nonpolar regions, aiding in fat digestion.

❖ The initial and rate-limiting step of bile acid synthesis is oxidation of cholesterol to 7α-hydroxycholesterol by a mixed function oxidase from the cytochrome P450 superfamily, cholesterol 7α- hydroxylase (CYP7A1).

❖ The synthesis of bile salts is under very tight regulatory control, mainly by a feedback mechanism on cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthetic pathway.

References

Chiang JYL. Regulation of bile acid synthesis. Front Biosci 1998;3:D176–93. 

Pullinger CR, Eng C, Salen G, et al. Human cholesterol 7α-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype. J Clin Invest 2002; 110(1):109–17. 

Russell DW. The enzymes, regulation, and genetics of bile acid synthesis. Annu Rev Biochem 2003;72:137–74. 

Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev 2003;83(2):633–71.

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