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Metabolism – Carbohydrates

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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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A 50-year-old, alcoholic male presents with a swollen face, distended abdomen, and an enlarged fatty liver. Fatty acids react with glycerol-3-P to form triglycerides, which accumulate to cause fatty liver. The liver has glycerol kinase, while adipose tissue lacks glycerol kinase. As a result, in adipose tissue, which of the following occurs?

A) Glucose cannot be converted to DHAP (dihydroxyacetone phosphate)

B) Glycerol cannot be converted to Glycerol-3-P

C) DHAP cannot be converted to Glycerol-3-P

D) Diacylglycerol cannot be converted to Triacylglycerol

E) Triacylglycerols cannot be stored.

The correct answer is- B) – Glycerol cannot be converted to Glycerol-3-P.

Glycerol kinase catalyzes the phosphorylation of glycerol to glycerol-3-p.

Glycerol released through adipolysis (breakdown of triglycerides) cannot be reutilized, as it has to be in the phosphorylated form, and glycerol kinase deficiency in adipose tissue makes glycerol a waste product (figure).

Therefore Glycerol is transported to liver, where upon conversion to dihydroxy acetone phosphate, (figure), it is either converted to glucose (through pathway of gluconeogenesis), or is completely oxidized through glycolytic pathway. The fate of glycerol is decided by the cellular requirements.

1)  In the fasting state- Glycerol released from lipolysis of adipose tissue triacylglycerol is used solely as a substrate for gluconeogenesis in the liver and kidneys. The conversion of glycerol to glucose requires phosphorylation to glycerol-3-phosphate by glycerol kinase and dehydrogenation to Dihydroxyacetone phosphate (DHAP) by glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (Figure). The G3PDH reaction is the same as that used in the transport of cytosolic reducing equivalents into the mitochondrion for use in oxidative phosphorylation. This transport pathway is called the glycerol-phosphate shuttle.

Fate of glycerol

Figure-Glucose-Glycerol cycle. Glycerol released from adipocyte is used in the liver either for energy production or is utilized as a substrate for gluconeogenesis. Glycerol is initially converted to glycerol-3-P, in a reaction catalyzed by glycerol kinase. Subsequently glycerol-3-P is converted to Dihydroxy acetone-P (DHAP) by glycerol-3-p dehydrogenase. It is a reversible reaction. DHAP can then enter the pathway of glucose production. Glucose produced is transported back to adipocyte to complete the cycle. The entry of glucose in the adipocyte is by GLUT4 receptors that are regulated by Insulin.

2) In the well fed state- Glycerol upon conversion to DHAP in liver (as described above), is oxidized completely through the pathway of glycolysis. Glycolytic pathway is involved both for the utilization and production of glycerol-3-P.

It is noteworthy that glycerol-3-P in adipose tissue is obtained through glycolytic pathway (figure), and not by direct phosphorylation of glycerol (glycerol kinase is absent in adipose tissue).  In fact adipocytes require a basal level of glycolysis in order to provide them with DHAP as an intermediate in the synthesis of triacylglycerols.

As regards other options

A) Glucose cannot be converted to DHAP (dihydroxy acetone phosphate) – This in incorrect, Glucose can be converted to DHAP through glycolytic pathway (figure).

C) DHAP cannot be converted to Glycerol-3-P- This is also incorrect, DHAP is converted to Glycerol-3-P, in a reaction catalyzed by Glycerol-3-P dehydrogenase.

D) Diacylglycerol cannot be converted to Triacylglycerol- Diacyl glycerol can be converted to triacylglycerol. This is also not a correct option.

E) Triacylglycerols cannot be stored- Incorrect again, Triacylglycerols can be stored in an unlimited amount in the adipose cells.

Thus the most appropriate option is B) – Glycerol cannot be converted to Glycerol-3-P due to deficiency of glycerol kinase.

 

 

 

 

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A 14-year-old high school girl who is extremely conscious about her appearance has gone  near fasting for two days to fit in to a dress she intentionally brought a size smaller than her actual size for a dance party. Which of the following organs/tissues contributes to the glucose that is being synthesized through gluconeogenesis?

A) Spleen

B) Red blood cells

C) Skeletal muscle

D) Liver

E) Brain

The correct answer is- D) Liver.

Gluconeogenesis is the process of converting non-carbohydrate precursors to glucose or glycogen.

Gluconeogenesis meets the needs of the body for glucose when sufficient carbohydrate is not available from the diet or glycogen reserves. A supply of glucose is necessary especially for the nervous system and erythrocytes. Failure of gluconeogenesis is usually fatal.

Liver and kidney are the major gluconeogenic tissues.

Substrates of Gluconeogenesis

The major substrates are-

  1. The glucogenic amino acids,
  2. Lactate
  3. Glycerol, and
  4. Propionate.

These noncarbohydrate precursors of glucose are first converted into pyruvate or enter the pathway at later intermediates such as oxaloacetate and Dihydroxyacetone phosphate (figure-1).

Pathway of gluconeogensis

 

Figure-1- Reactions of gluconeogenesis. Three irreversible reactions of glycolysis are substituted by alternative reactions. Pyruvate carboxylase, Phospho enol pyruvate carboxy kinase, Fructose 1,6 bisphosphatase and glucose-6-Phosphatase enzymes are unique to pathway of gluconeogenesis. Lactate enters as pyruvate, glycerol as Dihydroxy acetone-phosphate, propionate as Succinyl co A, and the intermediates of TCA cycle distal to α-Keto glutarate are glucogenic. Acetyl co A  is not glucogenic but it is a positive modulator of pyruvate carboxylase enzyme.

Role of kidney

Although the liver has the critical role of maintaining blood glucose homeostasis and therefore, is the major site of gluconeogenesis, the kidney also plays an important role. During periods of severe hypoglycemia that occur under conditions of hepatic failure, the kidney can provide glucose to the blood via renal gluconeogenesis. In the renal cortex, glutamine is the preferred substance for gluconeogenesis.

Glutamine is produced in high amounts by skeletal muscle during periods of fasting as a means to export the waste nitrogen resulting from amino acid catabolism. The glutamine is then transported to the kidneys where the reverse reactions occur. Glutamate is first produced from hydrolysis of Glutamine by glutaminase, which is then further catabolized  liberating ammonia and producing α-ketoglutarate which can enter the TCA cycle and the carbon atoms diverted to gluconeogenesis via oxaloacetate.

Role of kidney in gluconeogenesis

Figure-2- Role of kidney in gluconeogenesis

This process serves two important functions. The ammonia (NH3) that is liberated spontaneously ionizes to ammonium ion (NH4+) and is excreted in the urine effectively buffering the acids in the urine. In addition, the glucose that is produced via gluconeogenesis can provide the brain with critically needed energy.

 

 

 

 

 

 

 

 

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During acute myocardial infarction, the oxygen supply to an area of heart is reduced forcing the cardiac muscle cells to switch to anaerobic oxidation. Under this condition the activity of which of the following enzymes is increased by the increasing concentration of AMP?

A.  Phosphofructokinase-1

B.  Pyruvate kinase

C.  Citrate synthase

D.  Lactate dehydrogenase

E.  Succinate dehydrogenase

The correct answer is- A) Phosphofructokinase-1.

Lactate dehydrogenase is not the right option, Although the level of Lactate dehydrogenase rises in circulation after myocardial injury but it is not the enzyme that is affected by the increasing concentration of AMP.

Basic concept

AMP, a marker of low energy state, is a positive allosteric modifier for Phosphofuctokinase-1,  which is a rate limiting enzyme of glycolysis. Phospho fructokinase is the “valve” controlling the rate of glycolysis.  

ATP is an allosteric inhibitor of this enzyme.

In the presence of high ATP concentrations, the Km for fructose-6-phosphate is increased, glycolysis thus “turns off.” ATP elicits this effect by binding to a specific regulatory site that is distinct from the catalytic site. AMP reverses the inhibitory action of ATP, and so the activity of the enzyme increases when the ATP/AMP ratio is lowered. In other words, glycolysis is stimulated as the energy charge falls, or the AMP concentration rises.

Phosphofructokinase-1 is also regulated by D-fructose-2,6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase-1 for the substrate fructose-6-phosphate. Stimulation of phosphofructokinase is also achieved by decreasing the inhibitory effects of ATP. Fructose-2,6-bisphosphate increases the net flow of glucose through glycolysis by stimulating phosphofructokinase-1 and, by inhibiting fructose-1,6-bisphosphatase, the enzyme that catalyzes this reaction in the opposite direction.

AMP and Fructose 2,6 Bisphosphate are thus positive allosteric modifiers of Phosphofructokinase-1 (PFK-I), whereas ATP and Citrate are negative modifiers (Figure-1)

Citrate inhibits phosphofructokinase by enhancing the inhibitory effect of ATP.

Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated.

 Regulation of PFK-1

Figure-1- Phosphofructokinase-1 is regulated primarily by the energy needs of the cells. During the low energy conditions, glycolysis is stimulated to compensate for the energy. Reverse occurs in the presence of excess ATP (high energy state), the glycolysis is turned off.

Acute Myocardial infarction

Myocardial infarction (MI) is the irreversible necrosis of heart muscle secondary to prolonged ischemia. This usually results from an imbalance of oxygen supply and demand. This usually results from plaque rupture with thrombus formation in a coronary vessel, resulting in an acute reduction of blood supply to a portion of the myocardium.
The reduction of blood supply causes the change of aerobic mode of glycolysis to anaerobic, as a result lactate is the end product of glycolysis.

Lactate dehydrogenase(LDH) catalyzes the reversible conversion of pyruvate and lactate.

 Lactate-fermentation

Figure-2- Reaction showing the interconversion of pyruvate and lactate.

The appearance of LDH in the circulation generally indicates myocardial necrosis. It begins to rise in 12 to 24 hours following MI, and peaks in 2 to 3 days, gradually dissipating in 5 to 14 days.

Measurement of LDH isoenzymes is necessary for greater specificity for cardiac injury. There are 5 isoenzymes (1 through 5). Ordinarily, isoenzyme 2 is greater than 1, but with myocardial injury, this pattern is “flipped” and 1 is higher than 2.

LDH-5 from liver may be increased with centrilobular necrosis from passive congestion with congestive heart failure following ischemic myocardial injury.

 As regards other options

B.  Pyruvate kinase- It catalyzes the conversion of phosphoenolpyruvate to pyruvate, the last step of glycolysis.  AMP affect the activity of this enzyme also, but this is not a rate limiting enzyme of Glycolysis.

C.  Citrate synthase-catalyzes the condensation of Acetyl co A with oxaloacetate to form Citrate, the first step of TCA cycle.

D.  Lactate dehydrogenase- catalyzes the interconversion of pyruvate and lactate.

E.  Succinate dehydrogenase  is an enzyme of TCA cycle, it catalyzes the conversion of succinate to fumarate.

Thus the most appropriate answer is Phosphofructokinase-1, which is affected by the rising concentration of AMP to stimulate the overall pathway of Glycolysis.

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Case details

A pregnant woman is able to transfer oxygen to her fetus because fetal hemoglobin has a greater affinity for oxygen than does adult hemoglobin. Why is the affinity of fetal hemoglobin for oxygen higher?

A. The tense form of hemoglobin is more prevalent in the circulation of the fetus.

B. There is less 2, 3-BPG in the fetal circulation as compared to maternal circulation.

C. Fetal hemoglobin binds 2, 3-BPG with fewer ionic bonds than the adult form.

D. The Bohr Effect is enhanced in the fetus.

E. The oxygen-binding curve of fetal hemoglobin is shifted to the right.

 

The right answer is -C. 

The enhanced uptake of maternal oxygen by fetal Hb is due to less binding of 2, 3 BPG with fetal Hb.

It is not due to more prevalence of tense form of fetal hemoglobin in the circulation.

It is also not due to less 2, 3 BPG in the fetal circulation, the Bohr Effect is not enhanced in the fetus and the oxygen -binding curve of fetal Hb is also not shifted to the right.

Basic concept

2,3-BPG, is  the most concentrated organophosphate in the erythrocytes.  It is synthesized from Glucose by Luebering-Rapoport pathway which is a diversion from the main glycolytic pathway.

Luebering-Rapoport pathway

In erythrocytes, the reaction catalyzed by phosphoglycerate kinase may be bypassed, to some extent by the reaction of bisphosphoglycerate mutase, which catalyzes the conversion of 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate, followed by hydrolysis to 3-phosphoglycerate and Pi, catalyzed by 2,3-bisphosphoglycerate phosphatase (Figure-1). This alternative pathway involves no net yield of ATP from glycolysis. However, it does serve to provide 2,3-bisphosphoglycerate, which binds to hemoglobin, decreasing its affinity for oxygen, and so making oxygen more readily available to tissues .

 RL Shunt

Figure-1- RL Shunt- Formation and breakdown  of 2,3-bisphosphoglycerate. 

Significance of 2,3 BPG

In Hb A (adult Hb) when 2, 3-BPG binds to deoxyhemoglobin, it acts to stabilize the low oxygen affinity state (T state) of the oxygen carrier, exploiting the molecular symmetry and positive polarity by forming salt bridges with lysine and histidine residues in the four subunits of hemoglobin (figure-2).

The R state, with oxygen bound to a heme group, has a different conformation and does not allow this interaction. By selectively binding to deoxyhemoglobin, 2, 3-BPG stabilizes the T state conformation, making it harder for oxygen to bind hemoglobin and more likely to be released to adjacent tissues.

States of Hb 

Figure-2- Oxygen binding and unloading. 2,3 BPG being negatively charged binds to positively charged Histidine and Lysine residues of hemoblobin, stabilizing the tight binding state (T) or low affinity state that promotes oxygen unloading.

2,3-BPG can help to  prevent tissue hypoxia in conditions where it is most likely to occur. Conditions of low tissue oxygen concentration such as high altitude (2,3-BPG levels are higher in those acclimated to high altitudes), airway obstruction, anemia or congestive heart failure will tend to cause RBCs to generate more 2,3-BPG in their effort to generate energy by allowing more oxygen to be released in tissues deprived of oxygen.

This release is potentiated by the Bohr effect in tissues with high energetic demands.

Fetal hemoglobin (HbF) exhibits a low affinity for 2, 3-BPG, resulting in a higher binding affinity for oxygen. This increased oxygen-binding affinity relative to that of adult hemoglobin (HbA) is due to HbF’s having two α/γ dimers as opposed to the two α/β dimers of HbA (figure-3).

 Adult and fetal Hb

Figure-3- Adult Hb and fetal Hb.

The positive histidine residues of HbA β-subunits that are essential for forming the 2, 3-BPG binding pocket are replaced by serine residues in HbF γ-subunits so 2; 3-BPG has difficulties in linking to the fetal hemoglobin, hence the affinity of fetal hemoglobin for O2 increases. That’s the way O2 flows from the mother to the fetus (figure-4).

 Flow of oxygen

Figure-4-Flow of oxygen from maternal Hemoglobin to fetal hemoglobin. Maternal Hb has low affinity for oxygen (due to bound 2,3 BPG) whereas the affinity of fetal Hb for oxygen is more due to unavailable binding sites for 2,3 BPG.

 

 

 

 

 

 

 

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An intern is scrubbing into a complicated surgery that is anticipated to last for 15 hours. In preparation, the intern has not eaten from the past 15 hours. After 30 hours of fasting which of the following is most important for maintenance of normal blood glucose?

A. Glycogenolysis

B. Gluconeogenesis

C. Triacylglycerol synthesis

D. Increase Insulin release

E. Decrease muscle protein break down.

 

The correct answer is- B- Gluconeogenesis.

Significance of glucose

Glucose is a universal fuel molecule required for all cell types especially brain cells and the cells lacking mitochondria.

The blood glucose concentration is maintained in a range of 60-100 mg/dl to provide a constant supply of glucose to all cells especially brain cells and the cells lacking mitochondria, the latter are totally dependent upon glucose for their energy needs. Brain cells can utilize ketone bodies alternatively under conditions of prolonged fasting/starvation but glucose remains the preferred source of energy.

Fuel molecules during starvation

During starvation the energy needs are fulfilled by three types of fuels, glucose, fatty acids and ketone bodies.

Fatty acids due to long hydrophobic chains can’t cross through the Blood brain barrier (BBB), thus cannot be utilized by the brain. Ketone bodies although being relatively polar can cross through the blood brain barrier, yet they are utilized only under conditions of glucose deprivation. After several weeks of starvation, ketone bodies become the major fuel of the brain.

The oxidation of fatty acids and ketone bodies takes place in the mitochondria; hence the cells lacking mitochondria are left with no alternative fuels options except glucose to utilize. Thus an optimum glucose concentration has to be maintained for the proper functioning of these cells.

The sources of glucose include

1)      Diet

2)      Glycogen degradation, and

3)      Gluconeogenesis

Under conditions of inadequate dietary supply, the blood glucose levels are maintained initially by glucose supply from the stores (degradation of glycogen), the glycogen stores get exhausted within 14-16 hours. Thereafter the glucose is synthesized from the non-carbohydrate precursors (gluconeogenesis) such as lactate (the waste from the muscle), glycerol (the waste from the adipose tissue), and carbon skeleton of glucogenic amino acids (mainly obtained from the breakdown of muscle protein). The intermediates of TCA cycle and propionyl co A also serve as substrates for gluconeogenesis. The constant supply of substrates of gluconeogenesis, sustain the life of an individual.

In the given situation, after 30 hours of fasting, the only source of glucose is gluconeogenesis. Glycogenolysis cannot be expected after 30 hours.

As regards other options

  • Triacylglycerol synthesis cannot take place during conditions of starvation. Starvation is a state of catabolism. Glucagon the predominant hormone of this state, promotes adipolysis. Triacylglycerol synthesis takes place in the well fed state, in the presence of Insulin.
  • Increased insulin release is also incorrect, maximum insulin release occurs in the well fed state to promote utilization of glucose and to build up the body stores from the surplus nutrients. Under conditions of prolonged fasting as stated above, the insulin release is minimal, there is maximum release of glucagon to maintain blood glucose homeostasis.
  • Based on the similar concept of maintaining blood glucose homeostasis, decrease muscle protein breakdown during fasting is also inappropriate; there is rather a need for the constant supply of substrates of gluconeogenesis. Muscle protein breakdown can provide the carbon skeleton of amino acids, which can be further utilized for glucose production. The decreased insulin to glucagon ratio, and the decreased availability of circulating substrates, make this period of nutritional deprivation a catabolic state, characterized by initial degradation of glycogen, followed by degradation of triacylglycerol and protein.

Blood Glucose homeostasis (Summary)

Nutritional Status Source of blood glucose Cells using glucose as a fuel Major fuel of the brain
Well fed state Dietary glucose All cells Glucose
Post absorptive state Hepatic glycogenolysis (mainly) and gluconeogenesis All  cells except liver and skeletal muscle.

Adipose cells use at a reduced rate.

Glucose
Fasting Hepatic  and renal gluconeogenesis and some hepatic glycogenolysis Brain cells and  the cells lacking mitochondria, Glucose and ketone bodies
Prolonged fasting / Starvation Gluconeogenesis (mainly) The cells lacking mitochondria Only ketone bodies

 

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An 18 month-old- child is left unattended while in the kitchen and ingests a small portion of rat poison found in the cupboard found under the sink. The ingredient fluoroacetate reacts with Oxaloacetate to form fluorocitrate. Which pathway of the body is inhibited by this poison?

A. Glycolysis

B. TCA cycle

C. Fatty acid oxidation

D. Fatty acid synthesis

E. HMP pathway

The right answer is -B) TCA cycle.

Fluoroacetate is structurally similar to acetate, which has an important role in cellular metabolism.

Fluoroacetate disrupts the citric acid cycle (also known as the Krebs cycle or TCA cycle- Tricarboxylic acid cycle) by combining with coenzyme A to form Fluoroacetyl CoA, which reacts with oxaloacetate in the presence of citrate synthase (host enzyme) to produce fluorocitrate (Figure-1).

 Activation of Fluoroacetate

Figure-1- Activation of fluoroacetate is brought by Citrate synthase, an enzyme of TCA cycle

Fluorocitrate binds very tightly to Aconitase, to inhibit its action, (Figure-2) thereby inhibiting the citric acid cycle as a whole. This is an example of suicidal inhibition.

 TCA cycle

Figure-2- TCA cycle and the action of Aconitase

The direct effect of Fluoroacetate is on TCA cycle, but indirectly Glycolysis and other pathways are also inhibited.

Effect on Glycolysis

This inhibition results in an accumulation of citrate in the blood. Citrate and fluorocitrate are allosteric inhibitors of phosphofructokinase-1 (PFK-1), a key enzyme in the breakdown of Glucose. As PFK-1 is inhibited cells are no longer able to metabolize carbohydrates, depriving them of energy.

Effect on fatty acid oxidation

Inhibition of TCA cycle inhibits fatty acid oxidation also due to non availability of oxidized coenzymes, and incomplete utilization of Acetyl co A. But these are late implications. TCA cycle is vital to life. Death occurs immediately after its suppression. Directly Fluoroacetate has no effect on enzymes of fatty acid oxidation.

 Similarly fatty acid synthesis and HMP pathway are not directly affected by Fluoroacetate.

 

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A 50-year-old female presents with severe abdominal pain. Her serum amylase and lipase levels are abnormally elevated and she is diagnosed with pancreatitis. Which linkage between glucose residues is cleaved by amylase?

A. α- 1, 4

B. α- 1, 6

C. β-1, 4

D. α 1, 2

E. β-1, 6

Answer- A) alpha – 1, 4 is the right answer.

Amylase acts on starch and glycogen to cleave α- 1, 4 glycosidic linkages.

Dietary polysaccharides (starch and glycogen) are digested first by salivary amylase to form dextrins that are further shortened by pancreatic amylase to form maltose. Maltose is further digested by maltase to form two glucose residues (figure-1).

Both starch and glycogen are polymers of glucose. The two main constituents of starch are amylose (13–20%), which has a nonbranching helical structure, and amylopectin (80–85%), which consists of branched chains composed of 24–30 glucose residues united by α-1,4 linkages in the chains and by α-1 ,6 linkages at the branch points (figure-2).

Glycogen, a more highly branched structure than amylopectin contains chains of 12–14 α-D-glucopyranose residues (in α-1, 4 glucosidic linkage) with branching by means of α-1, 6 glucosidic bonds (figure-2).

Amylase acts on alpha – 1, 4 glycosidic linkages only, the branch point containing α- 1, 6 linkage cannot be cleaved by amylase. Both starch and glycogen are substrates for amylase, having the structural similarities.

Oveview of digestion of carbohydrates

Figure-1- An overview of digestion of carbohydrates. Salivary digestion is limited due to shorter duration of stay of food in the oral cavity. The polysaccharides are mainly digested by Amylases (salivary and pancreatic, that are isoenzymes, they catalyze the same reaction, but differ from each other in their physical properties)

As regards other options

Cellulose a polymer of glucose consists of β-D-glucopyranose units linked by β-1, 4 bonds to form long, straight chains strengthened by cross-linking hydrogen bonds. Mammals lack any enzyme that hydrolyzes the, β-1, 4 bonds, and so cannot digest cellulose (figure-3).

α 1,2 linkage, more precisely- O-α-D-glucopyranosyl-(1,2)-β -D-fructofuranoside linkage is found in sucrose that is hydrolyzed by sucrase.

Β-1, 6 linkages are not common in nutrients.

Amylose, amylopectin and glycogen structures

Figure-2- Structure of Amylose, amylopectin and glycogen. Amylopectin and glycogen are structurally alike, except for the fact that glycogen is more branched than amylopectin. Both components of starch and glycogen contain alpha 1, 4 glycosidic linkages, hence they are ideal substrates for Amylase.

Comparison of  strcutures of starch, glycogen and cellulose

Figure-3- Comparison of structures of starch, glycogen and cellulose. Starch and glycogen are similar structurally whereas cellulose, despite being a polymer of glucose cannot be digested by human beings as it contains beta 1, 4 glycosidic linkages. Cellulose is digested by Cellulase enzyme.

 

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A 45-year-old morbidly obese woman has been attempting to lose weight using a low- carbohydrate diet. After 2 months of little success, she confides in her son that she does add glucose to her coffee in the morning and after dinner but feels only some of this will be absorbed and should not be the cause of her limited success. Her son, a medical student, states that glucose is almost completely absorbed from the gut. What type of transport does glucose utilize for gastro intestinal absorption?

A. Active- Carrier mediated, against the concentration gradient and energy dependent

B. Facilitated- Carrier mediated, down the concentration gradient

C. Passive- Down the concentration gradient

D. Active and facilitated

E. Passive and facilitated

The correct answer iso D- Active and facilitated.

There are two separate mechanisms for the absorption of monosaccharides in the small intestine- Active and facilitated transport. Active transport is an energy dependent carrier mediated transport that involves transport of glucose against the concentration gradient. The carrier protein in active transport has two binding sites, one for glucose and the other for sodium. Thus it is a sodium dependent transport. Sodium transport is down the concentration gradient whereas the glucose transport is against the concentration gradient. Glucose and galactose are absorbed by a sodium-dependent process. They are carried by the same transport protein (SGLT 1), and compete with each other for intestinal absorption.

The second mechanism, facilitated transport also requires a carrier protein to speed up the process, but the energy is not invested in this transport and the flow is down the concentration gradient. Other monosaccharides (mainly) including glucose (but to a lesser extent) are absorbed by carrier-mediated facilitated diffusion. Fructose is mainly transported by Facilitated transport using GLUT-5 transport. Because they are not actively transported, fructose and sugar alcohols are only absorbed down their concentration gradient, and after a moderately high intake, some may remain in the intestinal lumen, acting as a substrate for bacterial fermentation.

Glucose is a polar molecule; the passive diffusion across the intestinal membrane is very-very slow. Thus only a small amount of glucose is absorbed by a passive diffusion.

 Passive versus Active transport

Figure- Passive diffusion versus active transport

Summary of Glucose transport

Features Passive diffusion Facilitated diffusion Active transport
Concentration gradient Down the concentration gradient from high to low. Down the concentration gradient from high to low. Against a concentration gradient from low to high
Energy expenditure none none Energy expenditure is in the form of ATP
Carrier protein/ transporter Not required required required
Speed Slowest mode Fast Fastest mode

Clinical significance

  • In SGLT- 1 deficiency, glucose is left unabsorbed and gets excreted in feces. Galactose is also malabsorbed since it is transported through the same transporter.
  • SGLT- 2, a similar glucose transporter is present in the renal tubular cells; the filtered glucose is reabsorbed back by this transporter. In its deficiency, glucose is not reabsorbed back, and is lost in urine, causing glycosuria.

 

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A young infant, who was nourished with a synthetic formula, had a sugar in the blood and urine. This compound gave a positive reducing sugar test but was negative when measured with glucose oxidase (specific test for detection or estimation of Glucose). Treatment of blood and urine with acid (which cleaves glycosidic bonds) did not increase the amount of reducing sugar measured. Which of the following compounds is most likely to be present in this infant’s blood and urine?
A. Glucose
B. Fructose
C. Maltose
D. Sorbitol
E. Lactose

The right answer is fructose.

Reducing sugars are usually detected by Benedict’s reagent, which contains copper sulphate, sodium citrate and sodium carbonate. Sodium carbonate makes the medium alkaline. Copper sulphate furnishes Cu2+ ions and sodium citrate prevents the precipitation of cupric ions as cupric hydroxide by forming a loosely bound cupric- sodium –citrate complex which on dissociation gives a continuous supply of cupric ions.

Benedict’s test

Principle

Carbohydrates with free aldehyde or ketone groups have the ability to reduce solutions of various metallic ions. Reducing sugars under alkaline conditions tautomerise and form enediols. Enediols are powerful reducing agents. They reduce cupric ions to cuprous form and are themselves converted to sugar acids. The cuprous ions combine with OH- ions to form yellow cuprous hydroxide which upon heating is converted to red cuprous oxide.

Procedure:

Take 5 ml of Benedict’s reagent. Add 8 drops of carbohydrate solution. Boil over a flame or in a boiling water bath for 2 minutes. Let the solution cool down.

Interpretation

Benedict’s test is a semi quantitative test. The color of the precipitate gives a rough estimate of a reducing sugar present in the sample (figure-1)

Green color- Up to 0.5 g %(+)

Green precipitate -0.5-1.0 g %(++)

Yellow precipitate -1.0-1.5 g %(+++)

Brick red precipitate- >2.0 G% (++++)

Negative benedict's testPositive benedict's test

(-ve)                (+ve)

Figure– The positive test is given by reducing sugars. The color of the precipitate determines the rough estimate of the reducing sugar present in the given sample.

Fehling test is an alternative to Benedict’s test. It differs from Benedict’s test in that it contains sodium potassium tartrate in place of Sodium citrate and potassium hydroxide as an alkali in place of sodium carbonate in Benedict’s reagent. It is not a preferred test over Benedict’s test since the strong alkali present causes caramelisation of the sugars; hence it is less sensitive than Benedict’s reagent.

Positive Benedict’s test for urine signifies Glycosuria.

Glycosuria is a non-specific term. Glucosuria, lactosuria, galactosuria, pentosuria and fructosuria denote the presence of specific sugars in urine.

Causes of Glycosuria are:

a. Renal glycosuria

b. Diabetes mellitus

c. Alimentary glucosuria

d. Hyperthyroidism, hyperpituitarism and hyperadrenalism

e. Stress, severe infections, increased intracranial pressure

Lactosuria– in lactose intolerance

Galactosuria– in galactosemia

Fructosuria– in hereditary fructose intolerance

Pentosuria – in essential pentosuria

Examples of non-carbohydrate substances which give a positive Benedict’s reaction are:

a) Creatinine

b) Ascorbic acid

c) Glucuronates

d) Drugs: Salicylates, PAS and Isoniazid.

Glucose oxidase test is a specific enzymatic method for the determination and estimation of glucose present in a given sample. True glucose can be estimated by this method.

As regards other options

Glucose cannot be present since specific test is negative.

Sorbitol is non reactive to reduction test.

Maltose and lactose would have caused increase in the amount of reducing sugar upon acid hydrolysis.

Hence it is fructose which is reducing in nature but non reactive to glucose oxidase.

 

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For Metabolism of fructose Lecture-1

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Clinical significance

1) Hereditary fructose Intolerance

Biochemical defect- Hereditary fructose intolerance is caused by mutation in the gene encoding Aldolase B enzyme.

Inheritance- It is an autosomal recessive trait that is equally distributed between the sexes.

Clinical features

These patients are healthy and asymptomatic until fructose or sucrose (table sugar) is ingested (usually from fruit, sweetened cereal, or sucrose-containing formula).

Clinical features include-

  • Recurrent vomiting,
  • Abdominal pain, and
  • Hypoglycemia that may be fatal.
  • Older patients who survive infancy develop a natural avoidance of sweets and fruits early in life and as a result frequently are without any dental caries.

Long-term exposure to fructose can result in

  • Liver failure
  • Renal tubulopathy,
  • Growth retardation

Pathophysiology- A defect in the Aldolase B gene results in a decrease in activity that is 15 percent or less than that of normal. This results in a buildup of Fructose-1-P levels in the hepatocytes.

The hypoglycemia that results following fructose uptake is caused due to

a) Inhibition of glycogenolysis, by fructose-1-phosphate, interfering with the phosphorylase action; and

b) Inhibition of gluconeogenesis at the deficient aldolase step.

Since the rate of fructose phosphorylation by fructokinase is so high, intracellular levels of both ATP and inorganic phosphate (Pi) are significantly decreased. The drop in ATP concentration adversely affects a number of cellular events, such as:

i) A hyperuricemic condition as a result of an increase in uric acid formation- See the details below (Figure 1)

ii) Severe hepatic dysfunction may be a manifestation of focal cytoplasmic degeneration and cellular fructose toxicity.

iii) Renal tubular dysfunction-The cause remains unclear; patients with renal tubular dysfunction primarily present with a proximal tubular acidosis complicated by aminoaciduria, glucosuria, and phosphaturia.

Laboratory findings

  • Urine analysis for the presence of reducing sugar- Based on the thorough dietary history of an ill child, the most straightforward approach to diagnosis of fructose 1-phosphate Aldolase deficiency is to demonstrate the presence of a non–glucose-reducing sugar in the urine. This is readily accomplished with Clinitest. Then, if test results are positive, thin-layer chromatographic separation should be used for confirmation.
  • Urine metabolic screening results may also provide evidence of glucosuria, proteinuria, and aminoaciduria, all of which are part of renal Fanconi syndrome.
  • Plasma electrolyte levels are important to determine, because the renal tubular acidosis component of hereditary fructose intolerance (HFI) may significantly depress the total plasma bicarbonate level.
  • Obtain liver function test results to assess the degree of hepatocellular disease.

Treatment

i)  Complete elimination of all sources of sucrose, fructose, and sorbitol from the diet. With this treatment, liver and kidney dysfunction improve, and symptoms become milder, even after fructose ingestion, and the long-term prognosis is good.

ii) Hepatomegaly may require months to resolve.

Complications

 Prolonged delay in diagnosis may result in cirrhotic changes with subsequent degeneration of function.

2) Essential Fructosuria

Biochemical defect- Essential fructosuria, also known as hepatic fructokinase deficiency or keto hexokinase deficiency, is a hereditary metabolic disorder caused by a deficiency of hepatic fructokinase, leading to fructose being excreted in the urine.

Inheritance– The inheritance is autosomal recessive.

Clinical manifestations

Essential fructosuria, apparently a harmless condition, is an extremely rare error of metabolism, the recognition of which is of importance because it may be mistaken for diabetes mellitus.It is characterized by the patient’s inability to utilize fructose normally, whether it is ingested as simple fructose or as a substance capable of yielding fructose on digestion, such as cane sugar. It is manifested clinically by a symptom less excretion of fructose in the urine.

Essential fructosuria should not be confused with hereditary fructose intolerance which is a very serious condition, and is due to deficiency of Aldolase B enzyme. It causes a rise in uric acid, growth abnormalities, in severe cases hepatic or renal failure and finally coma or death.

On the other hand, being symptomless, Fructosuria is commonly left undetected or undiagnosed.

3) Hyperuricemia upon excessive fructose ingestion

Fructose is mainly metabolized through Fructose-1-p pathway. Unlike phosphofructokinase, which is involved in glucose metabolism, fructokinase has no negative feedback system to prevent it from continuing to phosphorylate its substrate, i.e. Fructose to form fructose -1 phosphate, and as a consequence ATP can be depleted(Figure –1). ADP formed from the first step is converted to AMP, which is a substrate for AMP deaminase enzyme for further metabolism. 

Fructose induced hyperuricemia

Figure-1-The phosphorylation of fructose catalyzed by fructokinase is faster than subsequent cleavage by Aldolase, as a consequence ATP pool is depleted, with the resultant rapid degradation of ADP to AMP and finally to uric acid through intermediate formation of IMP, hypoxanthine and uric acid.

AMP deaminase enzyme that causes conversion of AMP to IMP (Inosine monophosphate) is regulated by inorganic phosphate. The rising concentration of inorganic phosphate inhibits this enzyme to prevent degradation of AMP. Upon excessive fructose ingestion, inorganic phosphate pool is depleted as a result the inhibition of AMP deaminase is lost. The overactive AMP deaminase converts AMP to IMP at an enhanced rate.  IMP, is subsequently converted to hypoxanthine then to xanthine and finally to uric acid. Excessive uric acid generation leads to gout or renal stones.

 AMP deaminase

Figure 2- AMP deaminase is inhibited by normal cellular concentrations of Pi. When these levels drop, the inhibition is released and AMP is converted to IMP and, ultimately to uric acid, thus excess uric acid is formed upon excessive fructose ingestion.

4) Obesity upon excessive fructose consumption

Excessive fructose intake (>50 g/d) has been found to be one of the underlying etiologies of obesity, insulin resistance and metabolic syndrome. The mechanism responsible for the metabolic changes may be described as follows-

Synthesis of triglycerides – Carbons from dietary fructose are found in both the free fatty acid and glycerol moieties of plasma triglycerides. Fructose undergoes more rapid glycolysis in the liver than does glucose, because it bypasses the regulatory step catalyzed by phosphofructokinase (Figure-3). This allows fructose to flood the pathways in the liver. High fructose consumption can lead to excess pyruvate production, causing a buildup of glycolytic intermediates and Acetyl co A.  Dihydroxyacetone phosphate (DHAP), the glycolytic intermediate, can be converted to glycerol 3-phosphate providing the glycerol backbone for the triglyceride molecule.  Excess Acetyl co A is channeled towards fatty acid synthesis.

Triglycerides , thus synthesized are incorporated into very low density lipoproteins (VLDL), which are released from the liver destined toward peripheral tissues for storage in both fat and muscle cells. Excessive fatty acid and triglyceride levels form the basis for the development of the metabolic syndrome, hypertension, glucose intolerance and type 2 diabetes mellitus.

In addition, unlike glucose, fructose does not stimulate insulin secretion or enhance leptin production. Because insulin and leptin act as key afferent signals in the regulation of food intake and body weight, this suggests that dietary fructose may contribute to increased energy intake and weight gain. Furthermore, calorically sweetened beverages may enhance caloric over consumption. Thus, the increase in consumption of HFCS (High fructose corn syrup) has a temporal relation to the epidemic of obesity, and the over consumption of HFCS in calorically sweetened beverages may play a role in the epidemic of obesity.

 Fructose induced obesity

Figure-3- Hypertriglyceridemia upon excessive fructose consumption

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Digestion and absorption of fructose

Fructose exists in foods as either a monosaccharide (free fructose) or as a unit of a disaccharide (sucrose). Free fructose is absorbed directly by the intestine; however, when fructose is consumed in the form of sucrose, digestion occurs entirely in the upper small intestine. As sucrose comes into contact with the membrane of the small intestine, the enzyme sucrase catalyzes the cleavage of sucrose to yield one glucose unit and one fructose unit. Fructose is absorbed in the small intestine, then enters the hepatic portal vein and is directed toward the liver.

Fructose absorption occurs on the mucosal membrane via facilitated transport involving GLUT5 (figure-1) transport proteins. Since the concentration of fructose is higher in the lumen, fructose is able to flow down a concentration gradient into the enterocytes, assisted by transport proteins. Fructose may be transported out of the enterocytes across the basolateral membrane by either GLUT2 or GLUT5, although the GLUT2 transporter has a greater capacity for transporting fructose and therefore the majority of fructose is transported out of the enterocytes through GLUT2 (figure-1).

Absorption of fructose

Figure-1- Absorption and transportation of fructose.

Clinical significance

Fructose Malabsorption- Fructose malabsorption, formerly named “dietary fructose intolerance,” is a digestive disorder in which absorption of fructose is impaired by deficient fructose carriers (GLUT 5) in the small intestine’s enterocytes. This results in an increased concentration of fructose in the entire intestine. In the large intestine, fructose that hasn’t been adequately absorbed exerts osmotic pressure, reduces the absorption of water and is metabolized by normal colonic bacteria to organic acids and the gases such as hydrogen, carbon dioxide and methane. This abnormal increase in hydrogen is detectable with the hydrogen breath test. The presence of gases and organic acids in the large intestine causes gastrointestinal symptoms such as bloating, diarrhea, flatulence, and gastrointestinal pain.

Restricting dietary intake of free fructose and/or fructose containing nutrients provides symptomatic relief in a high proportion of patients.

Metabolism of fructose

Much of the ingested fructose is metabolized by the liver, using the fructose 1-phosphate pathway (Figure-2).

1) The first step is the phosphorylation of fructose to fructose 1-phosphate by fructokinase.

2) Fructose 1-phosphate is then split into glyceraldehyde and dihydroxyacetone phosphate, the intermediates of glycolysis. This aldol cleavage is catalyzed by a specific fructose 1-phosphate aldolase.

3) Glyceraldehyde is then phosphorylated to glyceraldehyde 3-phosphate, a glycolytic intermediate, by triose kinase.

Metabolism of fructose

Figure-2- Fructose metabolism by Fructose-1-P pathway

4) Alternatively, fructose can be phosphorylated to fructose 6-phosphate by hexokinase. However, the affinity of hexokinase for glucose is 20 times as great as it is for fructose.

5) Fate of triose phosphates:

a) The two triose phosphates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, may be oxidized by glycolysis or

b) May condense together in the presence of Aldolase to form Fructose 1, 6 bisphosphate, that may be cleaved by fructose 1, 6 bisphosphatase to  fructose-6-P.

c) Glucose-6-P  produced from Fructose-6-P by the action of Phosphohexose isomerase

i)  May be hydrolyzed to free glucose by the action of glucose-6-phosphatase, or

ii) May enter HMP pathway for the production of NADPH and pentoses, or

iii) May be converted to Glucose-1-P to be used for glycogenesis (figure-3).

Thus glucose can be produced from fructose and can contribute to blood sugar levels.

Fate of trioses

Figure-3- Interrelation of fructose and glucose metabolism. There are two isoforms of Aldolase- Aldolase A and B. Aldolase A catalyzes the cleavage of fructose 1, 6 bisphosphate in glycolytic pathway, to form two phosphorylated trioses whereas Aldolase B, concerned with fructose metabolism catalyzes the cleavage of fructose-1-P to form Glyceraldehyde and Dihydroxyacetone -P. Glyceraldehyde has to be subsequently phosphorylated for further metabolism. The fate of trioses (oxidized or used for glucose production) depends upon the cellular conditions.

To be continued….

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1) A 30-year-old man has been fasting for religious reason for several days.  His brain has reduced its need for glucose by using which of the following substances as an alternate source of energy?

A. Fatty acids

B. Beta hydroxy butyrate

C. Glycerol

D. Beta carotene

E. Alanine

2) A 7-year-old girl is brought to the emergency department by her parents with complaints of severe polyuria and polydipsia. Laboratory examination reveals ketones in her urine. Which of the followings is the most likely source of ketones?

A. Fatty acid breakdown

B. Protein break down

C. Glycogenolysis

D. Gluconeogenesis

E. Side chain of cholesterol

3) A breast-fed infant began to vomit frequently and lose weight. Several days later she developed jaundice, hepatomegaly and bilateral cataract. What is the possible cause for these symptoms? 

A. Galactosemia

B. Von-Gierke’s disease

C. Juvenile diabetes Mellitus

D. Hereditary fructose intolerance

E.  Gaucher disease

4) The major metabolic product produced under normal circumstances by erythrocytes and by muscle cells during intense exercise is recycled through liver in the Cori cycle. The metabolite is-

A. Oxaloacetate

B. Alanine

C. Glycerol

D. Lactate

E. NADH

5) A 3-month-old infant presents with hepatosplenomegaly and failure to thrive. A liver biopsy reveals glycogen with an abnormal, amylopectin like structure with long outer chains and missing branches.  Which of the following enzymes would most likely be deficient?

A. Alpha Amylase

B. Branching enzyme

C. Debranching enzyme

D. Glycogen phosphorylase       

E. Glucose-6-phosphatase          

6) Prior to a race, many marathon runners will try to increase their glycogen concentrations by loading up with foods with high starch content, such as pasta. Alpha amylase secreted by the pancreas will digest the starch into which of the following major products?

A. Amylose, amylopectin, and maltose

B. Glucose, galactose, and fructose

C. Glucose, sucrose, and maltotriose

D. Limit dextrins, maltose, and maltotriose

E. Maltose, glucose and fructose

7) Which of the following substrates cannot contribute to net Gluconeogenesis in mammalian liver?

A. Alanine

B. Glutamate

C. Palmitate

D. Pyruvate

E. Odd chain fatty acids

8) Which of the following complications is less likely to occur in type II diabetics, as opposed to type I diabetics?

A. Retinopathy

B. Weight gain

C. Cardiovascular disease

D. Hypoglycemic coma

E. Non ketotic hyperosmolar coma

9) Familial fructokinase deficiency causes no symptoms because

A. Hexokinase can phosphorylate fructose

B. Liver Aldolase can metabolize it

C. Excess fructose does not escape in to urine

D. Excess fructose is excreted through feces

E. Excess fructose is converted to glucose

10) Which of the followings generates free glucose during the enzymatic breakdown of glycogen in skeletal muscles?

A. Phosphorylase

B. α-1-6-amyloglucosidase

C. Debranching enzyme

D. Glucose-6-phosphatase

E. Alpha amylase

1)   The answer is –B- Beta hydroxy Butyrate, a ketone body. Ketone bodies serve as alternative fuel for brain during prolonged fasting or starvation. Fatty acids due to long hydrophobic chain cannot cross blood brain barrier. Glycerol is a substrate of gluconeogenesis. In fact during prolonged fasting this is the only substrate left to provide glucose through pathway of gluconeogenesis. It can also be oxidized through glycolysis upon phosphorylation. Beta carotene is a provitamin; it is not a source of energy. Alanine is a transporter of amino group of amino acids from the muscle (glucose-alanine cycle), but it cannot be used as an alternative source of energy.

 2)   The answer is –A. Fatty acid break down provides Acetyl co A that serves as a precursor for ketone bodies. In Diabetes Mellitus glucose utilization is impaired due to absolute or relative insulin deficiency. Fatty acid breakdown occurs to provide energy and the resultant excessive Acetyl co A enters the pathway of ketogenesis. Protein breakdown provides amino acids, 6 amino acids are ketogenic, while 14 are glucogenic. Hence protein breakdown contributes only a little towards formation of Acetyl co A. The major contribution is through fatty acid breakdown. Glycogenolysis and Gluconeogenesis produce glucose only. Side chain of cholesterol provides propionyl co A which is a glucogenic component; it is converted to succinyl co A to gain entry in to TCA cycle.

 3)   The answer is –A. Galactosemia. The clinical manifestations are typical of classical Galactosemia. Bilateral cataract rules out the possibility of Von Gierke’s disease and hereditary fructose intolerance, although other symptoms are there in both these diseases. In juvenile diabetes mellitus, jaundice and hepatomegaly are not observed. In Gaucher disease, hepatomegaly is observed but cataract is never there.

 4)   The answer is-D- Lactate, the end product of glycolysis in erythrocytes and during intense exercise in skeletal muscle, is mobilized through Cori cycle to liver to provide glucose by the process of gluconeogenesis. (Erythrocytes lack mitochondria so the end product of glycolysis is always lactate.  The mode of glycolysis during intense exercise is anaerobic; hence lactate is formed as a result of glycolysis.

Alanine is transported to liver through Glucose Alanine cycle. Glycerol is also similarly transported but not from the erythrocytes or skeletal muscles, rather from the adipose tissues. Glycerol is a waste product in adipose tissues since without phosphorylation it cannot be utilized and the phosphorylating enzyme glycerol kinase is absent in adipose tissues.

 NADH produced at the step of glyceraldehyde dehydrogenase step is regenerated in the oxidized form NAD+ by reduction of pyruvate to lactate. These two reactions are coupled to have a continuous supply of NAD+.

 5)   The answer is-B- Branching enzyme. During the process of glycogen synthesis, branching enzyme creates branch points and further elongation is carried out by Glycogen synthase. In its deficiency stored glycogen is abnormal in chemistry, in the form of long polysaccharide chains with few branch points, resembling the structure of Amylopectin, thus this defect is also called Amylopectinosis. Alpha Amylase is an enzyme for digestion of starch and glycogen. Debranching enzyme deficiency results in the accumulation of abnormal glycogen, There is inability to remove the branch points, the resultant structure resembles Limit dextrin, and thus it is also called Limit dextrinosis. Glucose-6-phosphatase deficiency is observed in Von-Gierke’s diseases, a type 1 glycogen storage disease, the stored glycogen is always normal in chemistry.

 6)   The answer is-D.  The hydrolysis of starch is catalyzed by salivary and pancreatic amylases, which catalyze random hydrolysis of alpha (1- 4) glycoside bonds, yielding dextrins, and further hydrolysis yields a mixture of glucose, maltose, isomaltose (from the branch points in amylopectin) and maltotriose. Sucrose, galactose and fructose are not components of starch.

 7)   The answer is- C. Palmitate, a fatty acid with 16 carbon atoms, is not a substrate of gluconeogenesis. Even chain fatty acids, predominantly present in our body, yield Acetyl co A upon oxidation, which can not contribute towards gluconeogenesis.  The Pyruvate to Acetyl co A conversion is irreversible and moreover both of the carbon atoms of Acetyl co A are lost in the TCA cycle in the form of CO2.Oddchain fatty acid do act as substrates of gluconeogenesis, since propionyl co A the product of their oxidation can enter TCA cycle through formation of Succinyl co A, hence can contribute towards Glucose production. Alanine, pyruvate and glutamine are glucogenic.

 8)   The answer is-D- Hypoglycemic coma occurs as a result of insulin over dosage in Type 1diabetes Mellitus.  It is not observed in Type 2 diabetes. Weight gain can occur in both types, it is the result of treatment with insulin or certain hypoglycemic drugs. Non ketotic hyperosmolar coma is a frequent complication of coma especially in the elderly group.

 9)   The answer is-A Hexokinase is a non specific enzyme, it can phosphorylate fructose as well as other sugars but it has high km (low affinity) for fructose.  Glucose is the true substrate for this enzyme.Fructose-6-phosphatethe end product of Hexokinase reaction can enter glycolytic pathway to be utilized further, so it does not accumulate to produce the toxic effects. Liver Aldolase (Aldolase B) cleaves Fructose-1-P only, the product of fructokinase catalyzed reaction.  Aldolase A, present in all the cells of the body cleaves Fructose 1, 6 bisphosphate, the product of PFK-1 catalyzed reaction of glycolysis. Fructose to glucose conversion takes place only in the phosphorylated form.

 10) The answer is-B- Free glucose is released by the action of α-1-6-amyloglucosidase enzyme, a component of debranching enzyme. Debranching enzyme has two components. α-[1 4] to α-[1 4] Glucan transferase and α-1-6-amyloglucosidase.Glucan transferase shifts the trisaccharide on a branch bound by α-[1- 4] linkage to the straight chain and joins by α-[1 4] linkage. The exposed branch point is hydrolyzed by α-1-6-amyloglucosidase enzyme. Both components are present on the same polypeptide chain. Glucose-6- phosphatase does produce free glucose but it is absent in skeletal muscles. Alpha amylase is a digestive enzyme; it has no role in glycogen degradation in the muscle.

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Students can revise the concepts of glycogen metabolism through this power point presentation.

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1) Name the pathways that can utilize Glucose-6-P as a substrate.

2) What is the net energy output when the R/L shunt is active in the red blood cells?

3) Name an enzyme that uses inorganic phosphate for the phosphorylation of the substrate.

4) How is excessive fructose consumption related to obesity?

5) Why do sucrase and isomaltose deficiencies coexist?

6) Which pathway of glucose utilization is concerned with the production of ribose?

7) What is the biochemical basis of mental retardation in classical galactosemia?

8) What is the purpose of branching in glycogen structure?         

9) Glucose-6-P dehydrogenase deficiency primarily affects the red blood cells, why are the other cells of the body not affected by this deficiency?

10) Name the important inhibitors of glycolysis.

11) What is the significance of rising concentration of glucose-6-P on phosphorylase enzyme?

12) What is the normal range of blood glucose level?

13) Give example of substrate level phosphorylation in TCA cycle.

14) What is mechanism of inhibition of Aconitase enzyme by fluoroacetate?

15) How do odd chain fatty acids which are considered to be glucogenic gain entry in to the main pathway?

16) What is the major outcome of uronic acid pathway?

17) How many ATP molecules are produced when glucose is completely oxidized in skeletal muscle under aerobic conditions?

18) Name the key regulatory enzyme of glycogen synthesis.

19) How do skeletal muscles contribute towards maintenance of blood glucose level?

20) What is the cause of lactic acidosis in PDH complex deficiency?

21) The skeletal muscles are deficient in glucose-6-P dehydrogenase enzyme, how do they manage to synthesize ribose?

22) Name the concerned pathway for each of the following enzymes

a) Transaldolase

b) Phosphofructokinase-2

c) UDP-G pyro phosphorylase

d) Debranching enzyme

e) Aldolase B

23) Mention the role of 2,3 BPG in unloading of oxygen in a fetus ?

24) State the inhibitor of each of the following enzymes

a) Succinate dehydrogenase

b) Lactate dehydrogenase

c) Pyruvate dehydrogenase

d) Phospho triose isomerase

e) Enolase

25) Name the allosteric modifiers (negative and positive) for each of the following enzymes-

a) PFK-1

b) Fructose 1,6 bisphosphatase

c) Glycogen synthase

d) Phosphorylase

e) Pyruvate kinase

26) Name the enzyme catalyzing the following biochemical reactions-

a) Lactate to Pyruvate

b) Malate to oxaloacetate

c) Pyruvate to Alanine

d) Glucose-6-P to Fructose-6-P

e) Glyceraldehyde-3-P to Dihydroxyacetone –P

27) “Muscle glycogen phosphorylase is non responsive to glucagon”, what is the reason?

28) Phosphorylase enzyme is active in the dephosphorylated form, is it true or false?

29) What are the major tissues for gluconeogenesis?

30) Name the coenzyme for pyruvate carboxylase enzyme .

Note- Students should refer theory notes or subjective questions of carbohydrate metabolism for getting the answers.

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