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Case Studies

Laboratory results for a patient with uncontrolled Type I diabetes mellitus reveal hyperglycemia (456 mg/dL) and hypertriglyceridemia (395 mg/dL). The most likely cause of the hypertriglyceridemia in this patient is which of the following?

A. Deficiency in apoprotein C-II

B. Increased hepatic triglyceride synthesis

C. Decreased lipoprotein lipase activity

D. Deficiency in LDL receptors

E. Absence of hormone-sensitive lipase.

 

The correct answer is-C- Decreased Lipoprotein lipase activity.

Circulating lipoproteins are just as dependent on insulin as is the plasma glucose, it is because, the lipoprotein lipase that catalyzes the degradation of circulating lipoproteins is activated by insulin.

Lipoprotein lipase

Occurrence

Lipoprotein lipase is located on the walls of blood capillaries, anchored to the endothelium by negatively charged proteoglycan chains of Heparan sulfate. It has been found in heart, adipose tissue, spleen, lung, renal medulla, aorta, diaphragm, and lactating mammary gland, although it is not active in adult liver.

Lp L

Figure-1- Action of lipoprotein lipase (LpL).

It is not normally found in blood; however, following injection of heparin, lipoprotein lipase is released from its heparan sulfate binding into the circulation.  Due to this reason ‘heparin’ is called a “clearing factor”, as it causes release of lipoprotein lipase that promotes utilization and clearance of lipoproteins from the plasma.

Activators and inhibitors of lipoprotein lipase

Both phospholipids and apo C-II are required as cofactors for lipoprotein lipase activity (figure-1), while apo A-II and apo C-III act as inhibitors. Hydrolysis takes place while the lipoproteins are attached to the enzyme on the endothelium.

Hypertriglyceridemia in Type 1 Diabetes Mellitus

Dyslipidemia is a common metabolic abnormality in uncontrolled diabetes mellitus. Dyslipidemia includes; hypercholesterolemia, hypertriglyceridemia, raised LDLc and LDLc, but low levels of HDLc.

Biochemical basis of dyslipidemia in uncontrolled DM

One major role of insulin is to stimulate the storage of food energy following the consumption of a meal.This energy storage is in the form of glycogen in hepatocytes and skeletal muscle. Additionally, insulin stimulates synthesis of triglycerides in hepatocytes and promotes their storage in adipose tissue. In opposition to increased adipocyte storage of triglycerides is insulin-mediated inhibition of lipolysis (insulin inhibits adipolysis because the enzyme hormone- sensitive lipase, that catalyzes adipolysis is stimulated by Glucagon and inhibited by Insulin).

Lipolysis and its implications in Type 1 DM

In uncontrolled IDDM since the lipolysis is promoted, thus  there is a rapid mobilization of triglycerides leading to increased levels of plasma free fatty acids. The free fatty acids are taken up by numerous tissues (however, not the brain) and metabolized to provide energy. Free fatty acids are also taken up by the liver.

Increased fatty acid oxidation

Normally, the levels of malonyl-CoA are high in the presence of insulin. These high levels of malonyl-CoA inhibits carnitine acyl Transferase I, the enzyme required for the transport of fatty acyl-CoA’s into the mitochondria where they are subject to oxidation for energy production. Thus, in the absence of insulin, malonyl-CoA levels fall and transport of fatty acyl-CoA’s into the mitochondria increases. Mitochondrial oxidation of fatty acids generates acetyl-CoA which can be further oxidized in the TCA cycle.

Suppressed TCA cycle and its implications

a) Ketosis

In hepatocytes the majority of the acetyl-CoA is not oxidized by the TCA cycle but is metabolized into the ketone bodies, Acetoacetate and β-hydroxybutyrate.These ketone bodies leave the liver and are used for energy production by the brain, heart and skeletal muscle. In IDDM, the increased availability of free fatty acids and ketone bodies exacerbates the reduced utilization of glucose furthering the ensuing hyperglycemia. Production of ketone bodies, in excess of the body’s ability to utilize them leads to ketoacidosis. In diabetics, this can be easily diagnosed by smelling the breath. A spontaneous breakdown product of acetoacetate is acetone which is volatilized by the lungs producing a distinctive odor.

b) Hypercholesterolemia

The unutilized Acetyl Co A, due to suppressed TCA cycle, is also channeled towards the pathway of cholesterol biosynthesis resulting in hypercholesterolemia.

Basis of hypertriglyceridemia

Normally, plasma triglycerides are acted upon by lipoprotein lipase (LPL). In particular, LPL activity allows released fatty acids to be taken from circulating triglycerides for storage in adipocytes. The activity of LPL requires insulin and in its absence a hypertriglyceridemia results (figure-2).

Clinical pearls

In type 1 diabetes, moderately deficient control of hyperglycemia is associated with only a slight elevation of LDL cholesterol and serum triglycerides and little if any change in HDL cholesterol. Once the hyperglycemia is corrected, lipoprotein levels are generally normal. However, in obese patients with type 2 diabetes, a distinct “diabetic dyslipidemia” is characteristic of the insulin resistance syndrome. Its features are a high serum triglyceride level (300–400 mg/dL), a low HDL cholesterol (less than 30 mg/dL), and a qualitative change in LDL particles, producing a smaller dense particle whose membrane carries supranormal amounts of free cholesterol. These smaller dense LDL particles are more susceptible to oxidation, which renders them more atherogenic. Since low HDL cholesterol is a major feature predisposing to macro vascular disease, the term “dyslipidemia” has preempted the term “hyperlipidemia,” which mainly denoted the elevated triglycerides. Measures designed to correct the obesity and hyperglycemia, such as exercise, diet, and hypoglycemic therapy, are the treatment of choice for diabetic dyslipidemia, and in occasional patients in whom normal weight was achieved, all features of the lipoprotein abnormalities cleared.

As regards other options

Deficiency in apoprotein C-II

Apo CII is an activator of lipoprotein lipase, but its concentration is not decreased in diabetes mellitus.

Increased hepatic triglyceride synthesis

The Acetyl Co A carboxylase, the key regulatory enzyme of fatty acid biosynthetic pathway, is activated by insulin, thus, de novo fatty acid synthesis is decreased in insulin deficiency. However, the fatty acids mobilized from adipose tissue are used for esterification to form triglycerides which are transported from liver as VLDL. The excess flux of fatty acids that cannot be transported out as VLDL results in fatty liver. The hypertriglyceridemia in uncontrolled DM can be because of excess hepatic triglyceride synthesis, but mainly it is due to non degradation of circulating chylomicrons (carriers of dietary lipids) and VLDL (carriers of endogenous triglycerides) by the inactive lipoprotein lipase (figure-2).

Deficiency in LDL receptors

LDL receptors internalize LDL, their deficiency cannot cause hypertriglyceridemia, and otherwise also, they are not deficient in diabetes mellitus.

Absence of hormone-sensitive lipase

Hormone- sensitive lipase catalyzes the breakdown of triglycerides in adipose cells. It is activated by glucagon and catecholamines; inhibited by insulin. It is not absent; instead it is overactive in uncontrolled type 1 diabetes mellitus.

Hyper tgs 

 

Figure-2- Diabetic dyslipidemia. In normal health, VLDL released from liver, carrying endogenous triglycerides and Chylomicrons released from intestinal cells carrying dietary lipids, are acted upon by lipoprotein lipase (LPL), and the resultant fatty acids are taken up by peripheral cells, whereas the lipoprotein remnants are taken up by liver. VLDL is converted to LDL, through intermediate formation of IDL (intermediate density lipoprotein). In diabetes mellitus, in the absence of active LPL, the lipoprotein metabolism is disturbed resulting in hypertriglyceridemia, hypercholesterolemia, small dense LDL, low HDL and  a fatty liver.

 

 

 

 

 

 

 

 

 

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A 22- year-old diabetic comes to the Accident and Emergency department. She gives a 2-day history of vomiting and abdominal pain. She is drowsy and her breathing is deep and rapid. There is distinctive smell from her breath. She has been diagnosed with Diabetic ketoacidosis. Diabetic ketoacidosis is a complication of uncontrolled diabetes mellitus.

The TCA cycle in diabetes mellitus is suppressed and the excess Acetyl co A, resulting from fatty acid oxidation is channeled towards the pathway of ketogenesis.

Which of the following intermediates of TCA cycle is depleted in Type 1 Diabetes mellitus to suppress TCA cycle?

A) Succinate

B) Malate

C) α-Keto glutarate

D) Oxaloacetate

E) Pyruvate.

The correct answer is- D) – Oxaloacetate.

Two facts demand attention here-

1) TCA cycle suppression and

2) Basis of ketogenesis

In Diabetes mellitus, TCA cycle is in a state of suppression due to diminished availability of oxaloacetate which is channeled towards the pathway of gluconeogenesis.

The hyperglycemia in Insulin deficiency results from decreased utilization and excess pouring in of glucose. The processes of glucose utilization such as- Glycolysis, TCA cycle, HMP and glycogenesis occur at a diminished rate, whereas rates of gluconeogenesis and glycogen degradation are increased due to disturbed Insulin to Glucagon ratio in diabetes mellitus. Oxaloacetate is a common intermediate of  TCA cycle and gluconeogenesis. The utilization of oxaloacetate in the pathway of gluconeogenesis depletes the amount which is required for TCA cycle (Oxaloacetate acts as a catalyst; an optimum amount of oxaloacetate is required for the functioning of TCA cycle), therefore it undergoes in a state of suppression.

As glucose utilization is decreased in Diabetes mellitus, alternatively fatty acids are oxidized to compensate for the energy needs. Excess fatty acid oxidation results in:

i) Accumulation of NADH which further suppresses TCA cycle ( Excess of NADH decreases the catalytic activities of three NAD+ requiring enzymes of TCA cycle- Isocitrate dehydrogenase, Alpha ketoglutarate dehydrogenase and Malate dehydrogenase), and

Regulation of TCA cycle

Figure-1- Regulation of TCA cycle. Accumulation of NADH inhibits the activities of NAD + enzymes of TCA cycle, isocitrate dehydrogenase, Alpha keto glutarate dehydrogenase and Malate dehydrogenase. The activity of PDH complex is also decreased.

ii) Accumulation of Acetyl co A- The end product of fatty acid oxidation cannot be oxidized in TCA cycle at the same rate as that of its production, as a result , Acetyl co A is channeled either towards pathways of ketogenesis, or of cholesterol synthesis (figure-2).

TCA suppression

Figure-2- a) The rate of lipolysis is increased, fatty acids are oxidized to produce Acetyl CoA.

b) Due to non availability of oxaloacetate, which is diverted towards pathway of gluconeogenesis, TCA cycle is suppressed.

c) Acetyl co A is diverted towards pathway of ketogenesis. Acetone, acetoacetate and beta hydroxy butyrate are the three ketone bodies

d) Accumulated ketone bodies, (being acidic in nature and also as they deplete the alkali reserve) cause acidosis.

In Type 1 Diabetes mellitus, the onset of the disease is abrupt, which is why the body switches abruptly from glucose utilization to fatty acid oxidation for energy needs.  Acetyl co A resulting from excess fatty acid oxidation saturates TCA cycle and the other alternative pathways resulting in ketogenesis. This is the reason ketoacidosis is far more commonly found in type 1diabetes mellitus than type 2 diabetes.

The similar situation is observed in prolonged fasting or starvation. Diabetes mellitus and starvation depict a similar metabolic state, in both the conditions, the cells are deprived of glucose and switch to alternative fuels for their energy needs. The basis of ketosis is thus the same in both conditions.

As regards other options:

A) Succinate-Succinate is an intermediate of TCA cycle, but it is not depleted in Diabetes mellitus.

B) Malate- Similarly malate and C) α-Keto glutarate are also not depleted in Diabetes mellitus.

E) Pyruvate depletion does not directly affect the functioning of TCA cycle, of course pyruvate is also diverted towards glucose production, but there are other sources available, in any case TCA cycle activity is not affected.

Thus the most logical option is Oxaloacetate which is the most important regulator of TCA cycle, depletion of which suppresses TCA cycle.

 

 

 

 

 

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Case Details

A 40 -year-old man presents with chest pain that radiates to his left jaw and shoulder. He is diagnosed with a myocardial infarct (heart attack) and is prescribed a statin medication. Statins are competitive inhibitors of HMG CoA reductase, which converts HMG Co A to which of the following?

A. Isopentenyl pyrophosphate

B. Mevalonate

C. Geranyl pyrophosphate

D. Farnesyl pyrophosphate

E. Cholesterol.

The correct answer is B- Mevalonate.

Basic concept

Biosynthesis of cholesterol

The biosynthesis of cholesterol may be divided into five steps:

(1) Synthesis of Mevalonate from acetyl-CoA.

(2) Formation of Isoprenoid units from Mevalonate by loss of CO2.

(3) Condensation of six isoprenoid units forms Squalene.

(4) Cyclization of Squalene gives rise to the parent steroid, Lanosterol.

(5)Formation of cholesterol from lanosterol.

Always remember the formula

2+2= 4

4+2= 6

6-1=5

5+5=10

10+5=15

2×15=30

30-3=27

Details- (See the figure-1 and 2)

i) 2+2= 4

  • Initially, two molecules of acetyl-CoA (2+2) condense to form Acetoacetyl-CoA (4) catalyzed by cytosolic thiolase.

ii) 4+2=6

  • Acetoacetyl-CoA condenses with a further molecule of acetyl-CoA (4+2) catalyzed by HMG-CoA synthase to form HMG-CoA that is reduced to Mevalonate (6) by NADPH catalyzed by HMG-CoA reductase (figure-1).

Reaction catalyzed by HMG Co A reductase

Figure-1- Reaction catalyzed by HMG Co A reductase. HMG Co A reductase is inhibited by Statins by the mechanism of competitive inhibition and by bile acid, cholesterol and Mevalonate by feedback inhibition

Significance

  • The synthesis of Mevalonate is the committed step in cholesterol formation.
  • The enzyme catalyzing this irreversible step, 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), is an important control site in cholesterol biosynthesis,
  • It is the site of action of the most effective class of cholesterol-lowering drugs, the HMG-CoA reductase inhibitors (statins).

iii) 6-1=5

  • Decarboxylation (6-1 =5) yields Isopentenyl pyrophosphate (5), an activated isoprene unit that is a key building block for many important biomolecules (figure-2).

iv) 5+5=10

  • Isopentenyl pyrophosphate is isomerized by a shift of the double bond to form dimethylallyl pyrophosphate that condenses with another molecule of Isopentenyl pyrophosphate (5+5 =10) to form the ten-carbon intermediate Geranyl pyrophosphate (10).

v) 10+5=15

A further condensation with Isopentenyl pyrophosphate forms Farnesyl pyrophosphate (15). 

v) 2×15=30

Two molecules of Farnesyl pyrophosphate (15+15) condense at the pyrophosphate end to form Squalene (30)

vi) 30-3=27

  • Squalene can fold into a structure that closely resembles the steroid nucleus
  • Before ring closure occurs, Squalene is converted to Squalene 2, 3-epoxide by a mixed-function oxidase in the endoplasmic reticulum, Squalene peroxidase.
  • The methyl group on C14 is transferred to C13 and that on C8 to C14 as Cyclization occurs, catalyzed by oxidosqualene: lanosterol cyclase (figure-2).

The newly formed cyclized structure is Lanosterol.

  • The formation of cholesterol from lanosterol takes place in the membranes of the endoplasmic reticulum and involves changes in the steroid nucleus and side chain.
  • 3 carbon atoms are lost (30-3).
  • The double bond at C8–C9 is subsequently moved to C5–C6 in two steps, forming Desmosterol.
  • Finally, the double bond of the side chain is reduced, producing cholesterol.

 Steps of cholesterol synthesis

Figure-2- Steps of cholesterol biosynthesis

As regards other options

A. Isopentenyl pyrophosphate (IPP) – is formed from Mevalonate- PP

C. Geranyl pyrophosphate (C10) is formed by condensation of Isopentenyl pyrophosphate and Dimethyl allyl pyrophosphate

D. Farnesyl pyrophosphate (C15) is produced by condensation of Geranyl pyrophosphate and IPP

E. Cholesterol- is the end product of this pathway.

 

For further reading- Follow the link

http://www.slideshare.net/namarta28/cholesterol-synthesis-steps-and-regulation

 

 

 

 

 

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Case details

A 30- year-old pregnant woman has a sugar craving and consumes a hot fudge sundae. Her serum glucose level increases, which causes release of Insulin. Insulin is known to increase the activity of acetyl co A carboxylase, the rate limiting enzyme of fatty acid biosynthesis. Which of the following best describes this regulatory enzyme?

A. It is activated by carboxylation

B. It catalyzes a reaction that condenses an acetyl group with malonyl group

C. It catalyzes a reaction that requires biotin and ATP

D. It converts Malonyl co A to Acetyl co A

E. It is activated by malonyl Co A.

The correct answer is- C, It catalyzes a reaction that requires Biotin and ATP.

Basic concept

Acetyl co A carboxylase is the first enzyme of fatty acid biosynthesis that catalyzes the carboxylation of Acetyl co A to Malonyl CoA . The reaction catalyzed can be represented as follows :

 Reaction catalyzed by Acetyl co A carboxylase

Figure- Reaction catalyzed by Acetyl co A carboxylase. It is a carbon-carbon condensation, an energy requiring process.  ATP is the source of energy, and biotin is the coenzyme for this carboxylation process.

Acetyl co A carboxylase is the rate controlling enzyme in the pathway of lipogenesis. It is regulated by-

1) Allosteric modification-Acetyl-CoA carboxylase is an allosteric enzyme and is activated by citrate, which increases in concentration in the well-fed state and is an indicator of a plentiful supply of acetyl-CoA. Citrate converts the enzyme from an inactive dimer to an active polymeric form, with a molecular mass of several million. Inactivation is promoted by long-chain acyl-CoA molecules.

2) Feedback inhibition -The enzyme is inhibited by malonyl co A and palmitoyl co A, an example of negative feedback inhibition by a product of a reaction. Thus, if acyl-CoA accumulates because it is not esterified quickly enough or because of increased lipolysis or an influx of free fatty acids into the tissue, it will automatically reduce the synthesis of new fatty acid

3) Covalent Modification- Acetyl-CoA carboxylase is also regulated by hormones such as glucagon, epinephrine, and insulin via changes in its phosphorylation state

4) Induction and Repression-Insulin is an important hormone causing gene expression and induction of enzyme biosynthesis, and glucagon (via cAMP) antagonizes this effect. These mechanisms for longer-term regulation of lipogenesis take several days to become fully manifested. Prolonged consumption of high calorie or high carbohydrate diets causes an increase in acetyl co A carboxylase concentration by increasing the gene expression (induction). Conversely, a low-calorie diet or fasting causes a reduction in fatty acid synthesis by decreasing the synthesis of acetyl co A carboxylase (repression) .

Thus Insulin stimulates the synthesis of fatty acids by covalent modification and induction . It also provides glycerol-3-Phosphate through glycolytic pathway that can be used for esterification. That is the reason for weight gain in individuals on insulin therapy.

Based on the similar concept, obesity related with high carbohydrate diet can also be explained.

Excessive carbohydrate ingestion promotes triglyceride synthesis through following mechanisms-

1) Glycolysis yields pyruvate and hence Acetyl coA which is a precursor for fatty acid biosynthesis.

2) Glycolysis provides glycerol-3-p through dihydroxyacetone phosphate, that is used for esterification

3) HMP pathway provides NADPH which can be used for reductive biosynthesis.

All the pathways of glucose utilization are stimulated by Insulin and by these mechanisms, fatty acids are synthesized and esterified with glycerol to produce triglycerides. The adipose mass increases and the person becomes obese. That is the reason, low carbohydrate diet is recommended to those who want to lose weight.

As regards other options

Acetyl co A Carboxylase, the enzyme for fatty acid biosynthesis-

  • It is not activated by carboxylation
  • It  does not catalyze a reaction that condenses an acetyl group with malonyl group.
  • It does not convert Malonyl co A to Acetyl co A, instead Acetyl co A is converted to Malonyl co A .
  • It is  not activated by malonyl Co A. Malonyl co A, the product of this reaction inhibits this enzyme by feedback inhibition.

 

 

 

 

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Case details

An infant presents with lethargy, sweating, and irritability. He is admitted to the Pediatrics unit, where the attending pediatrician notices that his symptoms are pronounced when the feeding is delayed. After a series of tests, the child has been diagnosed with an enzyme deficiency that catalyzes the first step in the β-oxidation spiral, which is,

A. Fatty acid synthase

B. Acyl co A dehydrogenase

C. Enoyl Co A hydratase

D. β- Hydroxyacyl co A dehydrogenase

E. Thiolase

The correct answer is- Acyl co A dehydrogenase.

Acyl co A dehydrogenase is the first enzyme of β-oxidation spiral.

Basic concept

A saturated acyl Co A is degraded by a recurring sequence of four reactions (Figure)

1) Oxidation by flavin adenine dinucleotide (FAD)

2) Hydration,

3) Oxidation by NAD+, and

4) Thiolysis by Co A

The reaction catalyzed by acyl-CoA dehydrogenase  involves the removal of two hydrogen atoms from the 2( α)- and 3( β)-carbon atoms. The enzyme requires FAD as a coenzyme. (Figure).

Fatty acid oxidation is an important source of energy. Impaired activity of Acyl co A dehydrogenase or deficiency of any of the enzymes of beta oxidation leads to an energy deficit.

In the given case study, the infants presents with symptoms of hypoglycemia, due to an imbalance between demand and supply of glucose. In the conditions of impaired beta oxidation of fatty acids, glucose becomes the major source of energy, since glycogen stores are not well-developed and the substrates of gluconeogenesis are also not adequately available, the diet remains the only source of glucose. Any delay in feeding results in precipitation of symptoms of hypoglycemia.

Carnitine deficiency also produces hypoglycemia, due to similar reason of non- availability of energy from fatty acid oxidation (Carnitine acts as a transporter of fatty acids from cytoplasm to mitochondrial matrix since fatty  acids due to long hydrophobic chain cannot pass through inner mitochondrial membrane).

 

 Beta oxidation spiral

Figure- Steps of beta oxidation of fatty acids. The fatty acyl chain is shortened by two carbon atoms as a result of these reactions, and FADH2, NADH, and acetyl Co A are generated. Because oxidation is on the β carbon and the chain is broken between the α (2) – and β (3)-carbon atoms—hence the name – β oxidation.

As regards other options

Fatty acid synthase is a multienzyme complex involved in fatty acid synthesis.

Enoyl Co A hydratase- is an enzyme of beta oxidation (Figure); it catalyzes the second step of beta oxidation (hydration of fatty acids)

β- Hydroxyacyl co A dehydrogenase and Thiolase are also enzymes of beta oxidation spiral, but they catalyze the third and fourth steps (oxidation by NAD+ and thiolytic cleavage) respectively.

Thus the enzyme that catalyzes the first step in the β-oxidation spiral is Acyl co A dehydrogenase.

 

 

 

 

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A teenage girl was brought to the medical centre because of her complaints that she used to get too tired when asked to participate in gym classes. A consulting neurologist found muscle weakness in girl’s arms and legs. When no obvious diagnosis could be made, biopsies of her muscles were taken for test.

Biochemistry revealed greatly elevated amounts of triglycerides  esterified with primary long chain fatty acids. Pathology reported the presence of significant numbers of lipid vacuoles in the muscle biopsy

What is the probable diagnosis?

What is the cause for these symptoms?

Case details

The most likely cause of these symptoms is carnitine deficiency.

Carnitine deficiency

 The amino acid carnitine is required for the transport of long-chain fatty acyl coenzyme  esters into myocyte mitochondria, where they are oxidised for energy. Carnitine is obtained from foods, particularly animal-based foods, and via endogenous synthesis.

Carnitine deficiency results from inadequate intake of or inability to metabolize the amino acid carnitine. It can cause a heterogeneous group of disorders. Muscle metabolism is impaired, causing myopathy, hypoglycemia, or cardiomyopathy.Infants typically present with hypoglycemic, hypoketotic encephalopathy. Most often, treatment consists of dietary l-carnitine.

 

                      

Carnitine

 

Basic concept

Fatty acids are activated on the outer mitochondrial membrane, whereas they are oxidised in the mitochondrial matrix. The activation is brought by converting the fatty acid into Acyl co A ester under the activity of Acyl co A synthetase (1). A special transport mechanism is needed to carry long-chain acyl CoA molecules across the inner mitochondrial membrane.Activated long-chain fatty acids are transported across the membrane by conjugating them to carnitine. The acyl group is transferred from the sulphur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalysed by carnitine acyl transferase I (also called carnitine palmitoyl transferase I), which is bound to the outer mitochondrial membrane.(2)

Acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase. (3)

 The acyl group is transferred back to CoA on the matrix side of the membrane. This reaction, which is catalyzed by carnitine acyltransferase II (carnitine palmitoyl transferase II) (4),  is simply the reverse of the reaction that takes place in the cytosol.

Finally, the translocase returns carnitine to the cytosolic side in exchange for an incoming acyl carnitine (See figure).

 

Figure showing the role of carnitine in transporting the activated fatty acids in to the mitochondria.

Pathogenesis

A number of diseases have been traced to a deficiency of carnitine, the transferase or the translocase. The symptoms of carnitine deficiency range from mild muscle cramping to severe weakness and even death.The muscle, kidney, and heart are the tissues primarily affected. Muscle weakness during prolonged exercise is an important characteristic of a deficiency of carnitine acyl transferase because muscle relies on fatty acids as a long-term source of energy. Medium-chain (C8-C10) fatty acids, which do not require carnitine to enter the mitochondria, are oxidised normally in these patients. These diseases illustrate that the impaired flow of a metabolite from one compartment of a cell to another can lead to a pathological condition.

Causes of carnitine deficiency

Causes of carnitine deficiency include the following:

·        Inadequate intake (e.g., due to fad diets,lack of access, or long-term TPN)

·        Inability to metabolize carnitine due to enzyme deficiencies (e.g., carnitine palmitoyl Transferase deficiency.

·        Decreased endogenous synthesis of carnitine due to a severe liver disorder

·        Excess loss of carnitine due to diarrhoea,diuresis, or hemodialysis

·        A hereditary disorder in which carnitine leaks from renal tubules (Primary carnitine deficiency)

·        Increased requirements for carnitine when ketosis is present or demand for fat oxidation is high (eg, during a critical illness such as    sepsis or major burns; after major surgery of the GI tract)

·        Decreased muscle carnitine levels due to mitochondrial impairment

Primary Carnitine deficiency The underlying defect involves the plasma membrane sodium gradient–dependent carnitine transporter that is present in heart, muscle, and kidney. This transporter is responsible both for maintaining intracellular carnitine concentrations 20- to 50-fold higher than plasma concentrations and for renal conservation of carnitine.Primary carnitine deficiency has an Autosomal recessive pattern of inheritance.Mutations in the gene lead to the production of defective carnitine transporters. As a result of reduced transport function, carnitine is lost from the body and cells are not supplied with an adequate amount of carnitine.

Clinical manifestations of Carnitine deficiency

 Symptoms and the age at which symptoms appear depend on the cause. Carnitine deficiency may cause muscle necrosis, myoglobinuria, hypoglycemia, fatty liver, muscle aches, fatigue, and cardiomyopathy.

 1) The most common presentation is progressive cardiomyopathy with or without skeletal muscle weakness beginning at 2–4 yr of age.Energy deprived muscle cells are damaged.

 2) A smaller number of patients may present with fasting hypoketotic hypoglycemia during the 1st yr of life before the cardiomyopathy becomes symptomatic.Blockage of the transport of long chain fatty acids into mitochondria deprives the patient of energy production, as the fatty acid oxidation is impaired; all the energy needs are fulfilled by glucose oxidation. The resultant imbalance between demand and supply causes hypoglycemia. The compensatory ketosis in carnitine  induced hypoglycemia is not observed as the precursor, Acetyl co A is not available for ketone body production. The main source of Acetyl co is fatty acid oxidation  and that is impaired in carnitine deficiency.

3) Serious complications such as heart failure, liver problems, coma, and sudden unexpected death are also a risk.

Deficiencies in the Carnitine Acyl Transferase enzymes I and II can cause similar symptoms.

Diagnosis 

1)  Diagnosis of the carnitine transporter defect is aided by the fact that patients have extremely reduced carnitine levels in plasma and muscle (1–2% of normal).

2)  Fasting ketogenesis may be normal if liver carnitine transport is normal, but it may be impaired if dietary carnitine intake is interrupted.

3)  Hypoglycemia is a common finding. It is precipitated by fasting and strenuous exercise.

4) Muscle biopsy reveals significant lipid vacuoles.

Treatment

Treatment of this disorder with pharmacological doses of oral carnitine is highly effective in correcting the cardiomyopathy and muscle weakness as well as any impairment in fasting ketogenesis. All patients must avoid fasting and strenuous exercise. Some patients require supplementation with medium-chain triglycerides and essential fatty acids (eg, Linoleic acid, Linolenic acid). Patients with a fatty acid oxidation disorder require a high-carbohydrate, low-fat diet.

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Case Details

A 6-year- old child with progressive hearing loss wasbrought for consultation. History revealed that the child was born normal butprogressively developed loss of hearing and loss of smell.  From the past few months the child wasfinding it difficult to locate the things at night time.

The child had dysmorphic features, a flat bridge of nose,and low-set ears.

On examination, pulse was irregular and the liver was enlarged.Laboratory investigations revealed low levels of plasma cholesterol, HDL andLDL. A diagnosis of Refsum disease was made.

 What is the defectin this disease?

Refsum disease

Refsum disease (RD) is a neurocutaneous syndrome that is characterized biochemically by the accumulation of phytanic acid in plasma and tissues. Refsum first described this disease. Patients with Refsum disease are unable to degrade phytanic acid because of a deficient activity of Phytanic acid oxidase enzyme catalyzing the phytanic acid alpha-oxidation.

Peripheral polyneuropathy, cerebellar ataxia, retinitis pigmentosa, and Ichthyosis (rough, dry and scaly skin) are the major clinical components. The symptoms evolve slowly and insidiously from childhood through adolescence and early adulthood.

Biochemical defect

Refsum disease is an Autosomal recessive disorder characterized by defective alpha-oxidation of phytanic acid. Consequently, this unusual, exogenous C20-branched-chain (3, 7, 11, 15-tetramethyl hexadecanoicacid) fatty acid accumulates in brain, blood and other tissues. It is almost exclusively of exogenous origin and is delivered mainly from dietary plant chlorophyll and, to a lesser extent, from animal sources. Blood levels of phytanic acid are increased in patients with Refsum disease. These levels are 10-50 mg/dL, whereas normal values are less than or equal to 0.2 mg/dL, and account for 5-30% of serum lipids. Phytanic acid replaces other fatty acids,including such essential ones as Linoleic and Arachidonic acids, in lipid moieties of various tissues. This situation leads to an essential fatty acid deficiency, which is associated with the development of ichthyosis.

Reactions-

 

                                                  Phytanic acid

This process involves hydroxylation of the alpha carbon, removal of the terminal carboxyl group and concomitant conversion of the alpha hydroxyl group to a terminal carboxyl group, and linkage of CoA to the terminal carboxyl group. This branched substrate will function in the beta-oxidation process, ultimately yielding propionyl-CoA, acetyl Co As and, in the case of phytanic acid, 2-methyl propionyl CoA (Iso butyryl Co A).

 

 

Figure- showing steps of oxidation of phytanic acid. Pristanic acid is formed by alpha oxidation which subsequently undergoes beta oxidation to yield the final products.

Clinical manifestations

Refsum disease is rare, with just 60 cases observed so far.

Classic Refsum disease manifests in children aged 2-7 years; however, diagnosis usually is delayed until early adulthood.

Infantile Refsum disease makes its appearance in early infancy. Symptoms develop progressively and slowly with neurologic and ophthalmic manifestations. The disease is characterized by

·        Night blindness due to degeneration of the retina (retinitis pigmentosum)

·        Loss of the sense of smell (anosmia)

·        Deafness

·        Concentric constriction of the visualfields

·        Cataract

·        Signs resulting from cerebellar ataxia –

o   Progressive weakness

o   Foot drop

o   Loss of balance

·        Cardiac arrhythmias

·        Some individuals will have shortened bones in their fingers or toes.

·        The children usually have moderately dysmorphic features that may include epicanthal folds, a flat bridge of the nose,and low-set ears.

Laboratory Diagnosis

  • Levels of plasma cholesterol and high- and low-density lipoprotein are often moderately reduced.
  • Blood phytanic acid levels are elevated.
  • Cerebrospinal fluid (CSF) shows a protein level of 100-600 mg/dL.
  • Routine laboratory investigations of blood and urine do not reveal any consistent diagnostic abnormalities.
  • Phytanic oxidase activity estimation in skin fibroblast cultures is important

Imaging

Skeletal radiography is required to  estimate bone changes. 

Treatment

  • Eliminate all sources of chlorophyll from the diet.
    • The major dietary exclusions are green vegetables (source of phytanic acid) and animal fat (phytol).
    • The aim of such dietary treatment is to reduce daily intake of phytanic acid from the usual level of 50 mg/d to less than 5 mg/d.
  • Plasmapheresis – Patients may also require plasma exchange (Plasmapheresis) in which blood is drawn, filtered, and re infused back into the body, to control the buildup of phytanic acid.
    • The main indication for Plasmapheresis in patients with Refsum disease is a severe or rapidly worsening clinical condition.
    • A minor indication is failure of dietary management to reduce a high plasma phytanic acid level.

Prognosis 

Prognosis in untreated patients generally is poor. Dysfunction of myelinated nerve fibers and the cardiac conduction system leads to central and peripheral neuropathic symptoms, impaired vision, and cardiac arrhythmias. The latter frequently are the cause of death.

In early diagnosed and treated cases, phytanic acid decreases slowly, followed by improvement of the skin scaling and, to a variable degree, reversal of recent neurological signs. Retention of vision and hearing are reported.

Pharmacological up regulation of the omega-oxidation of phytanic acid may form the basis of the new treatment strategy for adult Refsum disease in the near future.

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Case details

Two sisters, aged 19 & 17 years, were referred to the dermatologist because they had large number of yellowish spots on the exposed parts of the body.

On thorough examination and after conducting a series of laboratory investigations they were advised to increase physical activity and reduce the intake of fats

What is the cause of yellow spots?

How are the medical advices going to help these patients?

Case Discussion

Both the sisters are having xanthomas, Xanthomas are lesions characterized by accumulation of lipid-laden macrophages. Xanthomas can develop in the setting of altered systemic lipid metabolism or as a result of local cell dysfunction. Most of the disorders of hyperlipidemia (Hyperlipoproteinemia) are associated with xanthomas.

Altered Lipoprotein metabolism (Hyperlipoproteinemia)

Lipoproteins

Lipids are insoluble in water; therefore, they are transported as complexes of lipoproteins with specific apoproteins. These proteins also serve as ligands to specific receptors, they facilitate transmembrane transport, and they regulate enzymatic activities. Lipoproteins may be classified according to their density, chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Lipoproteins may also be separated by electrophoresis into beta (LDL), prebeta (VLDL), Broad beta (IDL)and alpha (HDL) lipoproteins.

Metabolism of lipoproteins

The metabolic pathways of lipoproteins can be divided into exogenous and endogenous pathways. The exogenous lipoprotein pathway refers to the metabolism of intestinal lipoproteins, the triglyceride-rich chylomicrons, primarily formed in response to dietary fat.

The endogenous lipoprotein pathway refers to lipoproteins and apoproteins that are synthesized in tissues other than the intestines, predominantly in the liver. The liver secretes the triglyceride-rich VLDL that contains apoproteins B-100, C-II, and E into the circulation.

In the peripheral tissues, particularly adipose and muscle tissue, VLDL is cleaved by lipoprotein lipase (LPL), extracting most of the triglycerides and forming an IDL that contains apoproteins B-100 and E. IDL can be taken up by the liver through the LDL receptor, or it can be converted to the cholesterol-rich LDL that contains apoprotein B-100. LDL is removed from the circulation primarily by the liver through the LDL receptor.

 

Chylomicrons are similarly metabolized and are converted to chylomicron remnants after the action of lipoprotein lipase , which are internalized through remnant receptors (Apo E receptors) in to the liver.

The main role of HDL is to accept cholesterol and to transport it back to the liver (reverse cholesterol transport).

Lipoprotein (a) (Lp[a]) consists of an LDL-like particle with apoprotein B and a side chain of a highly glycosylated protein. Lp(a) has a role not only in atherogenesis but also in thrombogenesis because of its homology with plasminogen.

Hyperlipoproteinemia

Hyperlipoproteinemia is a metabolic disorder characterized by abnormally elevated concentrations of specific lipoprotein particles in the plasma. Hyperlipidemia (ie, elevated plasma cholesterol or triglyceride levels or both) is present in all hyperlipoproteinemia.

Hyperlipoproteinemia may be primary or secondary

Alterations in lipoproteins result either from genetic mutations that yield defective Apo lipoproteins (primary hyperlipoproteinemia) or from some other underlying systemic disorder, such as diabetes mellitus, hypothyroidism, or nephrotic syndrome (secondary hyperlipoproteinemia). The biochemical and genetic basis for the inherited disorders of lipid and lipoprotein metabolism differ considerably.

Primary Hyperlipoproteinemia

Traditionally, hyperlipidemia have been classified according to 5 phenotypes described by Fredrickson. These phenotypes are based on the Electrophoretic patterns of lipoprotein level elevations that occur in patients with hyperlipoproteinemia.

1) Type I hyperlipidemia

Familial lipoprotein lipase deficiency is an example of a primary disorder in which a deficiency of lipoprotein lipase in tissue leads to a type I pattern of hyperlipidemia, with a massive accumulation of chylomicrons in the plasma. This effect results in a severe elevation of plasma triglyceride levels. Plasma cholesterol levels are not usually elevated. Patients with type I may present in early childhood, often with acute pancreatitis. Eruptive xanthomas are the most characteristic skin manifestation of this disorder.(See figure below)

2) Type II hyperlipidemia

Cholesterol is bound to Apo lipoprotein B-100 as LDL in interstitial fluid. Cells may acquire cholesterol via an LDL receptor on the cell membrane. Familial LDL receptor deficiency and familial defective apoprotein B-100 are examples of primary defects that can lead to the accumulation of LDL, which corresponds to a type IIa pattern of hyperlipidemia. Plasma cholesterol levels are severely elevated, but plasma triglyceride levels are typically normal. Patients with type IIa have severe atherosclerosis and may present with tendinous or tuberous xanthomas as well as Xanthelasmas.(See figure below)

The type IIb pattern is characterized by the accumulation of both LDL and VLDL, with variable elevations of both triglyceride levels and cholesterol levels in the plasma. This is probably due to abnormal apo B protein.Patients with type IIb may present as adults with tendinous or tuberous xanthomas as well as Xanthelasmas.

3) Type III hyperlipidemia(Familial dysbetalipoproteinemia)

Type III hyperlipidemia is characterized by the accumulation of IDL, which is manifested by increases in both triglyceride levels and cholesterol levels in the plasma. A genetic basis for the primary disorder, familial dysbetalipoproteinemia, has been well established. Various mutations of apoprotein E impair its ability to bind to the IDL receptor. Patients with type III present as adults with premature atherosclerosis and xanthomas, particularly plane (palmar) xanthomas.(See figure below)

4) Type IV hyperlipidemia (Familial hypertriglyceridemia)

Familial hypertriglyceridemia is an example of a primary defect resulting in type IV hyperlipidemia.  there is over production of VLDL.Accumulation of VLDL causes severe elevations of plasma triglyceride levels. Plasma cholesterol levels are typically normal. A definitive molecular defect has not been established. Patients with type IV may present with eruptive xanthomas . This type of pattern is commonly associated with coronary heart disease, type II diabetes mellitus, obesity, alcoholism, and administration of progestational hormones.

5) Type V

Genetic defects of the apolipoprotein C-II gene result in the accumulation of chylomicrons and VLDL, which is the type V pattern of hyperlipidemia. Patients with this type have severe elevations of triglyceride levels in the plasma. These patients, like those with lipoprotein lipase deficiency, may present in early childhood with acute pancreatitis and eruptive xanthomas.

Other types of hyperlipoproteinemia

6) Decreased synthesis of HDL due to decreased formation of apoprotein A-I and apoprotein C-III leads to decreased reversed cholesterol transport, resulting in increased LDL levels, premature coronary artery disease, and plane xanthomas.

7) Hepatic lipase deficiency

Deficiency of the enzyme leads to accumulation of large triacylglycerol-rich HDL and VLDL remnants. Patients have xanthomas and coronary heart disease

Secondary Hyperlipidemia

Hyperlipidemia is also related to a variety of secondary causes. 

Secondary hypercholesterolemia can be found in pregnancy, hypothyroidism, cholestasis, and acute intermittent porphyria. 

Secondary hypertriglyceridemia can be associated with oral contraceptive use, diabetes mellitus, alcoholism, pancreatitis, gout, sepsis due to gram-negative bacterial organisms, and type I glycogen storage disease. 

Combined hypercholesterolemia and hypertriglyceridemia can be found in nephrotic syndrome, chronic renal failure, and steroid immunosuppressive therapy.

Xanthomas

Clinical manifestations

Cutaneous xanthomas associated with hyperlipidemia can be clinically subdivided into following types-

 

  • Xanthelasma palpebrarum is the most common of the xanthomas. The lesions are asymptomatic and usually bilateral and symmetric. The lesions are soft, velvety, yellow, flat, polygonal papules around the eyelids. Xanthelasma may be associated with hyperlipidemia. When associated with hyperlipidemia, any type of primary hyperlipoproteinemia can be present. Some secondary hyperlipoproteinemia, such as cholestasis, may also be associated with xanthelasmas.

 

 

 

 

 

 

 

 

 

Figure- showing Xanthelasma palpebrum

 

  • Tuberous xanthomas are firm, painless, red-yellow nodules. Tuberous xanthomas usually develop in pressure areas, such as the extensor surfaces of the knees, the elbows, and the buttocks. Tuberous xanthomas are particularly associated with hypercholesterolemia and increased levels of LDL. They can be associated with familial dysbetalipoproteinemia and familial hypercholesterolemia, and they may be present in some of the secondary hyperlipidemia (e.g., nephrotic syndrome, hypothyroidism).

 

 

 

 

 

 

 

 

 

 

 

Figure -showing Tuberous xanthomas

 

  • Tendinous xanthomas appear as slowly enlarging subcutaneous nodules related to the tendons or the ligaments. The most common locations are the extensor tendons of the hands, the feet, and the Achilles tendons. The lesions are often related to trauma. Tendinous xanthomas are associated with severe hypercholesterolemia and elevated LDL levels, particularly in the type IIa form. They can also be associated with some of the secondary hyperlipidemias, such as cholestasis.

 

 

 

 

 

 

 

 

 

 

 

Figure- showing tendinous xanthomas

 

  • Eruptive xanthomas most commonly arise over the buttocks, the shoulders, and the extensor surfaces of the extremities. The lesions typically erupt as crops of small, red-yellow papules on an erythematous base, and they may spontaneously resolve over weeks. Eruptive xanthomas are associated with hypertriglyceridemia, particularly that associated with types I, IV, and V (high concentrations of VLDL and chylomicrons). They may also appear in secondary hyperlipidemias, particularly in diabetes.

 

 

 

 

 

 

 

 

 

 

Figure showing eruptive xanthomas

 

  • Plane xanthomas can occur in any site. Involvement of the palmar creases is characteristic of type III dysbetalipoproteinemia. They can also be associated with secondary hyperlipidemia, especially in cholestasis. Generalized plane xanthomas can cover large areas of the face, the neck, and the thorax.

 

 

 

 

 

 

 

 

 

 

Figure showing plane xanthomas

 

Laboratory Investigations of hyper lipoproteinemia

  • Measurement of plasma lipid and lipoprotein levels while the patient is on a regular diet after an overnight fast of 12-16 hours. Abnormal lipoprotein patterns can often be identified after determining serum cholesterol and triglyceride levels and visual inspection of the plasma sample (stored at 4°C).
  • In some cases, performing electrophoresis and ultracentrifugation of whole plasma specimens may be necessary to help establish a diagnosis.
  • Appropriate blood, urine, and radiographic workups are required to rule out a secondary cause of hyperlipidemia. Lipoprotein profiles are primarily used to assess cardiac risk and to aid in the diagnosis of lipid metabolism disorders.

Treatment 

Treatment of the hyperlipidemia initially consists of diet and lipid-lowering agents such as statins, fibrates, bile acid–binding resins, probucol, or nicotinic acid. Xanthomas are not always associated with underlying hyperlipidemia, but when they are, diagnosing and treating underlying lipid disorders is necessary to decrease the size of the xanthomas and to prevent the risks of atherosclerosis.

Eruptive xanthomas usually resolve within weeks of initiating systemic treatment and tuberous xanthomas usually resolve after months, but tendinous xanthomas take years to resolve or may persist indefinitely.

 

Supportive care

  • Weight reduction and a diet low in saturated fat and cholesterol are advocated.
  • Patients should avoid alcohol and estrogen in certain types of hyperlipoproteinemia.

Prognosis- Prognosis is good if the underlying cause is treated.

 

 


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Case Details

A 45-year-old man presented to the emergency with chest pain. The chest pain lasted for approximately 15 minutes then subsided on its own. He also noticed that he was nauseated and was sweating during the pain episode. He had no medical problems and had not been to a physician for several years.

On examination, he was in no acute distress with normal vital signs. His lungs were clear to auscultation bilaterally, and his heart had a regular rate and rhythm with no murmurs. An electrocardiogram(ECG) revealed slight ischemic changes. The blood biochemistry revealed raised serum total cholesterol and LDL cholesterol levels.  He was placed on a low-fat diet and Lovastatin therapy.

He was without complaints and was feeling well on his subsequent follow-up visit. On repeat serum cholesterol screening, a decrease in the cholesterol level was noted.

What is the mechanism of action of this drug?

What are the potential side effects?

What are the alternative options to treat this patient?

Case discussion

The patients had an episode of IHD (Ischemic heart disease), and had hyperlipidemia.

Hyperlipidemia is one of the most treatable risk factors of coronary heart disease. Initially, when the fasting low-density lipoprotein (LDL) cholesterol is found elevated, life style modification is recommended such as dietary adjustments, exercise, and weight loss. If the LDL cholesterol level is again found above threshold, pharmacological therapy is initiated. Since the patient in the given case had a mild attack of IHD, hence without trial he had been put on low-fat diet and statins. 

Basic concept

A little more than half the cholesterol of the body arises by synthesis (about 700 mg/d), and the remainder is provided by the average diet. The liver and intestine account for approximately 10% each of total synthesis in humans. Virtually all tissues containing nucleated cells are capable of cholesterol synthesis, which occurs in the endoplasmic reticulum and the cytosol. LDL-C is a transporter of cholesterol from liver to peripheral tissues, while HDL is a transporter of cholesterol from peripheral tissues to liver for degradation. Excess LDL is responsible for Atherosclerosis and is a risk factor for IHD ( Ischemic heart disease), that is why it is considered “Bad cholesterol”. HDL-C on the other day acts as a scavenger to lower serum cholesterol level, because of this, it is cardio protective and is considered ‘Good cholesterol”.

Biosynthesis of cholesterol

The biosynthesis of cholesterol may be divided into five steps: (1) Synthesis of Mevalonate from acetyl-CoA. (2) Formation of isoprenoid units from Mevalonate by loss of CO2. (3) Condensation of six isoprenoid units form squalene. (4) Cyclization of squalene gives rise to the parent steroid, lanosterol. (5)Formation of cholesterol from lanosterol.

Hypercholesterolemia and the consequences

Atherosclerosisis characterized by the deposition of cholesterol and cholesteryl ester fromthe plasma lipoproteins into the artery wall. Diseases in which prolonged elevated levels of VLDL, IDL, chylomicron remnants, or LDL occur in the blood (e.g.,diabetes mellitus, lipid nephrosis, hypothyroidism, and other conditions of hyperlipidemia) are often accompanied by premature or more severe atherosclerosis. There is also an inverse relationship between HDL (HDL2) concentrations and coronary heart disease, making the LDL: HDL cholesterol ratio a good predictive parameter. This is consistent with the function of HDL in reverse cholesterol transport.

Diet Can Play an Important Role in Reducing Serum Cholesterol

Hereditary factors play the greatest role in determining individual serum cholesterol concentrations; however, dietary and environmental factors also play a part,and the most beneficial of these is the substitution in the diet of polyunsaturatedand monounsaturated fatty acids for saturated fatty acids. Plant oils such as corn oil and sunflower seed oil contain a high proportion of polyunsaturated fatty acids, while olive oil contains a high concentration of monounsaturated fatty acids. On the other hand, butter fat, beef fat, and palm oil contain a high proportion of saturated fatty acids. Sucrose and fructose have a greater effect in raising blood lipids, particularly triacylglycerols, than do other carbohydrates.

The reason for the cholesterol-lowering effect of polyunsaturated fatty acids is still not fully understood. It is clear, however, that one of the mechanisms involved is the up-regulation of LDL receptors by poly- and monounsaturated ascompared with saturated fatty acids, causing an increase in the catabolic rate of LDL, the main atherogenic lipoprotein. In addition, saturated fatty acids cause the formation of smaller VLDL particles that contain relatively more cholesterol, and they are utilized by extrahepatic tissues at a slower rate than are larger particles—tendencies that may be regarded as atherogenic.

Lifestyle and the Serum Cholesterol Level

Additional factors considered to play a part in coronary heart disease include high bloodpressure, smoking, male gender, obesity (particularly abdominal obesity), lack of exercise, and drinking soft as opposed to hard water. Premenopausal women appear to be protected against many of these deleterious factors, and this is thought to be related to the beneficial effects of estrogen. There is an association between moderate alcohol consumption and a lower incidence of coronary heart disease. This may be due to elevation of HDL concentrations resulting from increased synthesis of apo A-I. It has been claimed that redwine is particularly beneficial, perhaps because of its content of antioxidants.Regular exercise lowers plasma LDL but raises HDL.

Hypolipidemic Drugs

When diet changes fail, hypolipidemic drugs are prescribed to reduce Serum Cholesterol & Triacylglycerol levels. Few of the commonly used drugs to lower cholesterol level are as follows-

1) Statins (Lovastatin)

A family of drugs known as statins, have proved highly efficacious in lowering plasma cholesterol and preventing heart disease.

Mechanism of action of drug -Lovastatin is a member of a class of drugs (Atorvastatin, fluvastatin, pravastatin and Simvastatin are others in this class) called statins that are used to treat hypercholesterolemia. The statins act as competitive inhibitors of the enzyme HMG-CoA reductase.

These molecules mimic the structure of the normal substrate of the enzyme (HMG-CoA) and act as transition state analogues. While the statins arebound to the enzyme, HMG-CoA cannot be converted to mevalonic acid, thus inhibiting the whole cholesterol biosynthetic process. Effective treatment with Lovastatin, along with low fat diet, decreases levels of blood cholesterol. The lowering of cholesterol also lowers the amounts of the lipoproteins that transport cholesterol to peripheral tissues i.e. low density lipoproteins(LDL).

Side effects of therapy –The potential side effects include elevated liver function tests,increased muscle creatine phosphokinase (CPK) secondary to Myopathy and rarely rhabdomyolysis.

2) Alternative treatment options-Other agents that may be considered include bile acid sequestrants,Niacin, fibric acid, and fish oils.

a) Niacin is a vitamin that is used in high doses to treat hypercholesterolemia. Niacin acts to decrease VLDL and LDL plasma levels.Its mechanism of action is not clearly understood but probably involves inhibition of VLDL secretion, which in turn decreases the production of LDL. Niacin inhibits the release of free fatty acids from adipose tissue which leads to a decrease of free fatty acids entering the liver and decreased VLDL synthesis in the liver. This decreases the availability of VLDL for conversion to LDL (containing cholesterol esters). Niacin also increases high-densitylipoprotein (HDL) (the “good cholesterol”) by an unknown mechanism.

b) Fibrates such as Clofibrate and gemfibrozil act mainly to lower plasma triacylglycerols by decreasing the secretion of triacylglycerol and cholesterol-containing VLDL by the liver.

c) Ezetimibe– a new drug, Ezetimibe, which reduces blood cholesterol levels by inhibiting the absorption of cholesterol by the intestine, has recently been introduced. Ezetimibe belongs to the azetidinone class of cholesterol absorption inhibitors.

d) Bile Acid Sequestrants (Resins)-Bile acid sequestrants bind bile acids in the intestine and promote their excretion in the stool. To maintain the bile acid pool size, the liver diverts cholesterol to bile acid synthesis. The decreased hepatic intracellular cholesterol content results in up regulation of the LDL receptor and enhanced LDL clearance from the plasma. Bile acid sequestrants include Cholestyramine, colestipol, and colesevelam.

e) Omega 3 Fatty Acids (Fish Oils)-ω-3 polyunsaturated fatty acids (ω -3 PUFAs) are present in high concentration in fish and in flax seeds. The most widely used ω -3 PUFAs for the treatment of hyperlipidemia are the two active molecules in fish oil: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fish oils can result in an increase in plasma LDL-C levels in some patients. Fish oil supplements can be used in combination with Fibrates,niacin, or statins to treat hypertriglyceridemia. In general, fish oils are well tolerated and appear to be safe, at least at doses up to 3–4 g. A lower dose of omega 3 (about 1 g) has been associated with reduction in cardiovascular events in CHD (Chronic Heart Disease) patients and is used by some clinicians for this purpose.

Management of Low HDL-C

 Causes of low HDL levels

·        Severely reduced plasma levels of HDL-C(<20 mg/dL) accompanied by triglycerides <400 mg/dL usually indicate the presence of a genetic disorder, such as a mutation in apoA-I, LCAT(Lecithin Cholesterol Acyl Transferase) deficiency,or Tangier disease.
·        HDL-C levels <20 mg/dL are common in the setting of severe hypertriglyceridemia, in which case the primary focus should be on the management of the triglycerides.
·        HDL-C levels <20 mg/dL also occur in individuals using anabolic steroids.
·        Secondary causes of moderately low levels of plasma HDL (20–40 mg/dL) should be considered in conditions like smoking, Type 2 Diabetes mellitus, Gaucher’s disease and malnutrition.

Management

 1) Smoking should be discontinued.

2) Obese persons should be encouraged to lose weight, sedentary persons should be encouraged to exercise.

3) Diabetes should be optimally controlled. 

4) When possible,medications associated with reduced plasma levels of HDL-C should be discontinued. 

5)The presence of an isolated low plasma level of HDL-C in a patient with a borderline plasma level of LDL-C should prompt consideration of LDL lowering drug therapy in high-risk individuals. 

6) Statins increase plasma levels of HDL-C only modestly (~5–10%). 

7) Fibrates also have only a modest effect on plasma HDL-C levels (increasing levels ~5–15%), except in patients with coexisting hypertriglyceridemia, where they can be more effective. 

8) Niacin is the most effective available HDL-C–raising therapeutic agent and can be associated with increases in plasma HDL-C by up to ~30%, although some patients do not respond to niacin therapy.


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Case Details

A 26-year-old female at 32 weeks of gestation presented to the clinic with complaints of generalized itching. Patient reported no rash or skin changes. She denied any change in detergent, soaps, or perfumes. She denied nausea and vomiting .There was no history of any drug intake or previous allergies. There was no fever or any other medical illness.

On physical examination, there were no rashes apparent on her skin and only some excoriations were there from itching. 

Laboratory investigations revealed slightly elevated serum transaminases and bilirubin levels, Alkaline phosphatase levels were much higher than normal.

What is the patient’s likely diagnosis?

What is the cause of the patient’s generalized itching?

Case discussion

The patient is most probably having intrahepatic cholestasis ofpregnancy.

Generalized pruritus in pregnancy and a characteristic enzyme profile; it’s a typical picture of intrahepatic cholestasis of pregnancy. High alkaline phosphatase is a marker of cholestasis. (It is also high late in normal pregnancy due to the influx of placental alkaline phosphatase, But in cholestasis the level may be 4 times the normal reference range). Slightly high transaminases differentiate it from viral hepatitis in which very high levels are found and the high levels (In the present case) are due to bile salts induced toxic damage to the liver cells.Bilirubin is high due to intrahepatic obstruction as a result of cholestasis.  This is conjugated type. Increased serum bile salts and accumulation of bile salts in the dermis of the skin are responsible for generalized itching.

Intrahepatic cholestasis of pregnancy (ICP) is a benign disorder that occurs in the second or third trimester and resolves spontaneously after delivery. Cholestasis of pregnancy is a condition in which the normal flow of bile from the gallbladder is impeded, leading to accumulation of bile salts in the body. 

Basic concept of Bile Salts

Bile salt molecules secreted by the gallbladder are essential for the emulsification and absorption of fats. They are the salt forms of bile acids, which are the major product of cholesterol catabolism in the liver.

There are two primary bile acids formed in the liver from cholesterol: cholic acid and Chenodeoxycholic acid. The formation of bile acids prevents cholesterol accumulation in organs;  since the body cannot break down the steroid ring of cholesterol. Bile acids are conjugated with Glycine or taurine in the liver prior to secretion, forming glyco- or tauro conjugates. 

Bacterial enzymes present in the intestine produce the secondary bilesalts, deoxycholate and lithocholate, by reducing the primary bile salts. Bile salts perform important functions and are recycled by the body.In the gut, the glycine or taurine moieties are removed from the bile salts.They are reabsorbed in the small intestine along with  the secondary bile acids and returned to the liver for reuse via the portal vein.The body produces 400 mg of bile salts per day from cholesterol; this represents the fate of half of the cholesterol used daily in metabolism (800 mg). Between 20 to 30 g of bile salts secreted from the liver in to the duodenum per day,  less than 0.5 g per day of bile salts are lost through excretion.

 

Figure- showing enterohepatic recirculation of bile salts

Intrahepatic cholestasis of pregnancy is a syndrome of unknown etiology characterized by a 100-fold increase in maternal and fetal blood bile salt levels. Bile salts are produced in both the fetal and maternal liver. The fetus transfers the bile salts across the placenta for disposal. When the function of the maternal gallbladder is slowed, bile salts can accumulate in the liver and bloodstream, ultimately resulting in the classical pruritus symptoms. It is believed that pregnancy-related hormones may slow bile salt excretion from the gallbladder.

It is speculated that the hormones such as estrogen and progesterone, which are elevated in pregnancy, cause a slowing of the gallbladder function, leading to this disorder. The incidence of intrahepatic cholestasis of pregnancy is highest during the third trimester, when estrogen levels peak.

Symptoms

 Generalized pruritus is the main symptom of intrahepatic cholestasis of pregnancy. The itching may be more intense over the palms and soles but can extend to the trunk, extremities,eyelids, and, in rare cases, the oral cavity. The pruritus is worse at night.Jaundice is uncommon (10-25%); when it occurs, jaundice follows the onset of pruritus.

Diagnosis

 The serum bilirubin levelis usually lower than 6 times the upper limit of reference range and is usually conjugated. Fever is rare. Steatorrhea may be present, which may lead to deficiency of fat-soluble vitamins, especially vitamin K. Aminotransferases levels are less than 1000 U/L, which distinguishes intrahepatic cholestasis (IHC) from viral hepatitis. Alkaline phosphatase is 4 times higher than the reference range (44-147 U/L). Interestingly, gamma-glutamyltranspeptidase levels are within the reference range or only mildly elevated.Liver biopsy is rarely needed for diagnosis but reveals cholestasis with minimal hepatocellular necrosis.

 Treatment

Medical treatment is directed at relieving maternal symptoms and improving fetal outcome. Pruritus can be controlled with antihistaminics, and ursodeoxycholic acid.

More severe cases may require bile salt binders such as Cholestyramine or corticosteroids. Fetal outcome is improved with early diagnosis and prompt treatment.

The most successful therapy for cholestasis of pregnancy has been ursodeoxycholic acid.Ursodeoxycholic acid is a naturally occurring bile acid, which, when administered, relieves both pruritus and liver function abnormalities.Experimental evidence suggests that it protects hepatocytes from bile acid-induced cytotoxicity and improves hepatobiliaryexcretion. Additionally, it decreases bile salt transfer to the fetus and improves the secretory function of placental trophoblastic cells. Ursodeoxycholicacid is recycled through the enterohepatic circulation.

Cholestyramine is another treatment option for cholestasis of pregnancy. It is an oral medication that binds bile salts in the intestine and promotes their excretion in the feces. As this drug is not absorbed, it most likely has little effect on the fetus. Effects on the fetus are still under evaluation. However, Cholestyramine can interfere with the absorption of fat soluble vitamins, such as vitamins A, D, E, and K. In rare cases, drug-induced vitamin K deficiency is believed to contribute to hemorrhages during childbirth.


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