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Case Studies

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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Case details

A 3-year-old boy was brought to the emergency department after several episodes of vomiting and lethargy. His pediatrician was concerned about his failure to thrive and possible hepatic failure along with recurrent episodes of the vomiting and lethargy. After a careful history, it was observed, that these episodes occur after ingestion of certain types of food, especially high in fructose.

His blood sugar was checked in the emergency department and was extremely low (42 mg/dL). The test for reducing sugar in urine was positive.

What is the most likely diagnosis?

What is the biochemical basis for the clinical symptoms?

What is the treatment of the disorder?

Case Discussion

The child is most probably suffering from ‘Hereditary fructose Intolerance’. The onset of symptoms after ingestion of fructose or fructose containing diet is a sign of hereditary fructose Intolerance’. All these symptoms of vomiting, lethargy, failure to thrive, hypoglycemia and liver failure are characteristic of this disease.

Hereditary fructose Intolerance

Basic concept

In the liver, kidney, and intestine, fructose can be converted to glycolytic/ gluconeogenic intermediates by the action of three enzymes—fructokinase, aldolase B, and triokinase (also called triose kinase). In these tissues, fructose is rapidly phosphorylated to fructose 1-phosphate (F1P) by fructokinase at the expense of a molecule of adenosine triphosphate (ATP). This has the effect of trapping fructose inside the cell. A deficiency in this enzyme leads to the rare but benign condition known as essential fructosuria.

In other tissues such as muscle, adipose, and red blood cells, hexokinase can phosphorylate fructose to the glycolytic intermediate fructose 6-phosphate (F6P). Fructose- 1-phosphate is further metabolized to dihydroxyacetone phosphate (DHAP) and glyceraldehyde by the hepatic isoform of the enzyme Aldolase, which catalyzes a reversible aldol condensation reaction. Aldolase is present in three different isoforms. Aldolase A is present in greatest concentrations in the skeletal muscle, whereas the B isoform predominates in the liver, kidney, and intestine. Aldolase C is the brain isoform. Aldolase B has similar activity for either fructose 1,6-bisphosphate (F16BP) or F1P; however, the A or C isoforms are only slightly active when F1P is the substrate. Glyceraldehyde may be converted to the glycolytic intermediate, glyceraldehyde 3-phosphate (GAP), by the action of the enzyme triokinase. This enzyme phosphorylates glyceraldehyde at the expense of another molecule of ATP. The GAP can then enter into the glycolytic pathway and be further converted to pyruvate, or recombine with DHAP to form F16BP by the action of Aldolase.

 

 

 

 

 

 

 

 

 

 

 

Figure-1- The metabolic pathway for the entry of fructose into the glycolytic pathway. Fructokinase rapidly converts fructose to fructose 1-phosphate, which in the liver is cleaved by Aldolase B to dihydroxyacetone phosphate (DHAP) and glyceraldehyde.

Biochemical defect

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

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 F1P levels in the hepatocytes. Because the maximal rate of fructose phosphorylation by fructokinase is so high (almost an order of magnitude greater than that of glucokinase), intracellular levels of both ATP and inorganic phosphate (Pi) are significantly decreased. The drop in ATP concentration adversely affects a number of cellular events, including detoxification of ammonia, formation of cyclic AMP (cAMP), and ribonucleic acid (RNA) and protein synthesis. The decrease in intracellular concentrations of Pi leads to a hyperuricemic condition as a result of an increase in uric acid formation. 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, uric acid.

The toxic effects of F1P can also be exhibited in patients that do not have a deficiency in aldolase B if they are parenterally fed with solutions containing fructose. Parenteral feeding with solutions containing fructose can result in blood fructose concentrations that are several times higher than can be achieved with an oral load. Since the rate of entry into the hepatocyte is dependent on the fructose gradient across the cell, intravenous loading results in increased entry into the liver and increased formation of F1P. Since the rate of formation of F1P is much faster than its further metabolism, this can lead to hyperuricemia and hyperuricosuria by the mechanisms described above.

 

 

 

 

 

 

 

Figure-2- showing the inhibition of AMP deaminase by inorganic phosphate (Pi). A decrease in [Pi] increases the activity of AMP deaminase and leads to increased production of uric acid. Competition between urate and lactate for renal tubule excretion accounts for the lactic acidemia.

The cause of severe hepatic dysfunction remains unknown but may be a manifestation of focal cytoplasmic degeneration and cellular fructose toxicity. The cause of renal tubular dysfunction also remains unclear; patients with renal tubular dysfunction primarily present with a proximal tubular acidosis complicated by aminoaciduria, glucosuria, and phosphaturia. Thus, in an infant who is homozygous for fructose 1-aldolase deficiency, fructose ingestion triggers a cascade of biochemical events that result in severe clinical disease.  

Inheritance

Hereditary fructose intolerance is an autosomal recessive trait that is equally distributed between the sexes.

Age

In many infants, the age at symptom onset leads to the diagnosis. An accurate dietary history can indicate a link between the introduction of fruits into the diet and symptom onset.

Incidence

The incidence of hereditary fructose intolerance in the Caucasian population has been estimated at 1 in 20,000 births. Although the true prevalence has not been established, hereditary fructose intolerance may be more common than originally believed; many asymptomatic affected people may simply avoid the ingestion of most or all sweets.

Clinical Manifestations

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.
  • Progression of liver and kidney failure, eventually leading to death.

Laboratory findings

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.

Other Tests

Elimination of dietary fructose is both a compulsory and therapeutic step. In patients who are ill, elimination may also serve as a diagnostic test because all symptoms should completely resolve.

Only asymptomatic patients in a controlled setting should undergo intravenous fructose tolerance testing; use oral fructose tolerance testing is avoided because of the potentially violent GI response.

The combination of a therapeutic response to fructose elimination and a positive response to the fructose tolerance test is sufficient to exclude obtaining a biopsy sample. However, a molecular analysis in leucocytes of the gene on chromosome 9 may provide definitive evidence of a mutation at the q22.3 band.

Treatment

Consists of the complete elimination of all sources of sucrose, fructose, and sorbitol from the diet. With this treatment, liver and kidney dysfunction improve, and catch-up growth is common. Intellectual development is usually unimpaired. As the patient matures, symptoms become milder, even after fructose ingestion, and the long-term prognosis is good. Hepatomegaly may require months to resolve. Prolonged delay in diagnosis may result in cirrhotic changes with subsequent degeneration of function.

Medication

Drug therapy is not a component of the standard of care for this condition.

Mortality/Morbidity

Morbidity is implicit in untreated patients. Hypoglycemia and acidosis may act together to cause organ shock or coma. Ongoing hepatocellular insult may result in cirrhosis and eventual hepatic failure. Failure to thrive progressing to cachexia is the rule. Mortality may result from any or all of the above conditions

 

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Case details

A 2-year-old black girl was referred to the hematologist after her pediatrician found her to be severely anemic with splenomegaly and jaundice. Her mother gave a possible history of a “ blood problem” in her family but did not know for sure.

Her hemoglobin electrophoresis was normal, and the complete blood count (CBC) revealed a normocytic anemia. The platelet and white blood cell counts were normal. On the peripheral smear, there were many bizarre erythrocytes, including spiculated cells. A diagnosis of pyruvate kinase deficiency was made.

What is the underlying biochemical mechanism for this disorder?

How is this disorder inherited?

Case discussion

The girl has hemolytic anemia and jaundice due to pyruvate kinase deficiency.

Pyruvate Kinase Deficiency (Pkd)

Pyruvate kinase deficiency (PKD) is one of the most common enzymatic defects of the erythrocyte. This disorder manifests clinically as a hemolytic anemia, but surprisingly, the symptomatology is less severe than hematological indices indicate. Presumably, this is due to enhanced oxygen delivery as a result of the defect.

The clinical severity of this disorder varies widely, ranging from a mildly compensated anemia to severe anemia of childhood. Most affected individuals do not require treatment. Individuals who are most severely affected may die in utero of anemia or may require blood transfusions or splenectomy, but most of the symptomatology is limited to early life and to times of physiologic stress or infection.

Inheritance

Autosomal recessive.

Pathophysiology

Pyruvate kinase deficiency (PKD) is an erythrocyte enzymopathy involving the Embden-Meyerhof pathway of anaerobic glycolysis. Erythrocytes have evolved without oxidative phosphorylation to form adenosine triphosphate (ATP), the compound essential for providing the erythrocyte energy. A discrepancy between erythrocyte energy requirements and ATP-generating capacity produces irreversible membrane injury, resulting in cellular distortion, rigidity, and dehydration. This leads to premature erythrocyte destruction by the spleen and liver. Low ATP levels are responsible for erythrocyte intracellular electrolyte concentration disruption, due to failure of the adenosine triphosphatase cation pump (Na-K ATPase pump).

 

Fig. 1: Pathway of anaerobic glycolysis in RBCs

Basic Concept

As shown in Figure 1, two mols of ATP are formed per mole of glucose metabolized by the glycolytic cycle. The primary final product of glucose metabolism by glycolysis in the RBC is not pyruvate, as in other tissues most of the time, but lactate. Since RBCs have no mitochondria, NAD+ cannot be regenerated by shuttling NADH produced in glycolysis into the mitochondrial electron transport system. Therefore, the only option to continue glycolysis is to regenerate NAD+ from NADH by reducing pyruvate to lactate in the lactate dehydrogenase reaction. But lactate is not the sole product of RBC glycolysis. Methemoglobin reductase uses some of the NADH produced by glycolysis to reduce methemoglobin (Fe3+) back to active hemoglobin (Fe2+) capable of binding O2 for transport to the tissues. Thus, the final products are a mixture of lactate and pyruvate with lactate being the primary product.

In tissues other than the RBC, pyruvate has alternative metabolic fates that, depending on the tissue, include gluconeogenesis, conversion to acetyl-CoA by pyruvate dehydrogenase for further metabolism to CO2 in the tricarboxylic acid (TCA) cycle, transamination to alanine or carboxylation to oxaloacetate by pyruvate carboxylase.

In the RBC, however, the restricted enzymatic endowment precludes all but the conversion to lactate. The pyruvate and lactate produced are end products of RBC glycolysis that are transported out of the RBC to the liver where they can undergo the alternative metabolic conversions described above.

How does compromise of pyruvate kinase (PK) activity lead to anemia? Pyruvate kinase lies at the end of the glycolytic pathway in RBCs followed only by lactate dehydrogenase. Pyruvate kinase activity is critical for the pathway and therefore critical for energy production. If ATP is not produced in amounts sufficient to meet the energy demand, then those functions are compromised. Energy is required to maintain the Na+/K+ balance within the RBC and to maintain the flexible discoid shape of the cell. In the absence of sufficient pyruvate kinase activity and therefore ATP, the ionic balance fails, and the membrane becomes misshapen. Cells reflecting pyruvate kinase insufficiency rather than a change in membrane composition are removed from the circulation by the macrophages of the spleen. This results in an increased number of circulating reticulocytes and possibly bone marrow hyperplasia, which is a biological response to lowered RBC count as a result of hemolysis of erythrocytes.

The hexose monophosphate shunt and glutathione synthetic pathway protect the erythrocyte against destruction from free radicals and oxidative stress. Loss of adequate ATP diminishes their functions also.

Young reticulocytes retain mitochondria that produce ATP through oxidative phosphorylation. However, this comes at a price, a 6- to 7-fold higher oxygen requirement. Paradoxically, this can lead to the demise of any reticulocyte, because its journey through the spleen, an environment deficient in glucose and oxygen, is lengthened by its adhesive tendency. In such an environment, the reticulocyte is at an increased risk of metabolic failure.

Important intermediates proximal to the PK defect influence erythrocyte function. Two to three-fold increases of 2, 3-bisphosphoglycerate levels result in a significant rightward shift in the hemoglobin-oxygen dissociation curve. Physiologically, the hemoglobin of affected individuals has an increased capacity to release oxygen into the tissues, thereby enhancing oxygen delivery. Thus, for a comparative hemoglobin and Hematocrit level, an individual with PKD has an enhanced exercise capacity and fewer symptoms. This is particularly advantageous during pregnancy, because it enhances transfer of oxygen to the fetal blood. This most likely adds to the particularly benign course of this disease in many affected individuals. Women with PKD typically do not require transfusions during pregnancy.

There are two distinct genes encoding PK activity. One is located on chromosome 1 and encodes the liver and erythrocyte PK proteins (identified as the PKLR gene) and the other is located on chromosome 15 and encodes the muscle PK proteins (identified as the PKM gene). The muscle PKM gene directs the synthesis of two isoforms of muscle PK termed PK-M1 and PK-M2. Deficiencies in the PKLR gene are the cause of the most common form of inherited non spherocytic anemia.

Enzyme defects that have been described include decreased substrate affinity, increased product inhibition, decreased response to activator, and thermal instability.

Incidence

• Although pyruvate kinase deficiency (PKD) occurs worldwide, most cases have been reported in northern Europe, Japan, as well as in the United states

No sex preference has been detected for pyruvate kinase deficiency.

• The age of onset for inherited pyruvate kinase deficiency (PKD) correlates with severity. Persons with severe disease usually have onset in the neonatal period or infancy. In most affected persons, PKD is detected during childhood, but in individuals who are mildly affected, PKD may not be detected until late adulthood.

• Acquired PKD is usually secondary to a particular disease. In such cases, the age of onset varies with the primary disease.

Clinical Manifestations

• Birth history reveals—severe anemia, severe jaundice, kernicterus or history of exchange transfusion

• Anemia, mild to severe

– Growth delay

– Failure to thrive

– May become symptomatic during times of physiological stress, including acute illness, particularly viral, and pregnancy

• Family history consistent with autosomal recessive inheritance

• Frontal bossing

• Jaundice

• Abdomen—Splenomegaly mild to moderate, upper right quadrant tenderness, Murphy sign positive

• Extremities—Chronic leg ulcers

Causes

• Medical conditions, such as acute leukemia, preleukemia and refractory sideroblastic anemia, as well as complications from chemotherapy, can cause an acquired pyruvate kinase deficiency. This type is more common and milder than the hereditary type.

• More than 100 genetic defects of the PK gene have been detected. Most defects are missense mutations, but splicing mutations, insertions, and deletions also occur. Although inheritance is clinically autosomal recessive, most affected individuals are compound heterozygous for 2 different mutant alleles.

Treatment

In patients with mild to moderate disease, care is predominantly supportive in nature.

• Red blood cell transfusion may be necessary if the hemoglobin value falls significantly. This tends to occur in early childhood and during periods when physiologic stress is present, such as when an infection exists or during pregnancy.

• Bone marrow transplantation can be performed.

Surgical care—Splenectomy is indicated only for patients with severe anemia.

Prevention

• Monitor the Hematocrit value carefully during times of physiologic stress.

• If the defects in the parents are known, prenatal diagnosis using deoxyribonucleic acid (DNA) testing is possible.

Complications

• Cholecystolithiasis is common in the first decade of life for children with severe anemia.

• Splenectomy increases the risk of (1) sepsis by encapsulated bacteria for children and (2) thromboembolic disease for adults.

• Ischemic stroke has been reported in previously undiagnosed young adults with pyruvate kinase deficiency.

• Multiple-transfusion therapy can cause iron overload.

• Blood transfusions expose a person to the risk of contracting certain infections that are not well detected

(e.g. human immunodeficiency virus [HIV] disease, hepatitis C).

• Repeated transfusions during pregnancy increase the risk of alloimmunization, which may lead to fetal complications.

 Prognosis

• Mild and moderate forms of the disease are associated with an excellent prognosis.

• Severe forms of the disease are mostly symptomatic during early childhood. Following early childhood, the disorder is much better tolerated.

• Most morbidity develops from the above-mentioned complications.

• Hydrops fetalis has been reported in a severely affected fetus.

Things to remember

1. Pyruvate kinase deficiency (PKD) is an erythrocyte enzymopathy involving the Embden-Meyerhof pathway of anaerobic Glycolysis.

2. A discrepancy between erythrocyte energy requirements and ATP-generating capacity produces irreversible membrane injury, resulting in cellular distortion, rigidity, dehydration and premature erythrocyte destruction by the spleen and liver.

3. Although inheritance is clinically autosomal recessive; most affected individuals are compound heterozygous for 2 different mutant alleles.

4. In patients with mild to moderate disease, care is predominantly supportive in nature.

5. Splenectomy is indicated only for patients with severe anemia.

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Case details

A 12- year-old girl who had a grossly enlarged abdomen reported to OPD . She had a history of frequent episodes of weakness, sweating and pallor that were eliminated by eating. Her development had been slow; she sat at the age of 1 year, walked unassisted at the age of 2 years, and was doing poorly in the school.

Physical examination revealed normal blood pressure,temperature and a normal pulse rate but a sub normal weight (23 Kg).The liver was enlarged, firm and was descended in to pelvis. The spleen was not palpable,nor were the kidneys. The remainder of the physical examination was within the normal limits.

Laboratory investigation report revealed, low blood glucose, low p H, high lactate, triglycerides, ketones and high free fatty acids. The liver biopsy revealed high glycogen content. Hepatic glycogen structure was normal. The enzyme assay performed on the biopsy tissue revealed very low glucose-6- phosphatase levels.

What is the probable diagnosis?

What is the possible treatment for this patient?

Case details

The girl is suffering from Von –Gierke’s disease. The clinical picture, biochemical findings, hypoglycemia and increased Hepatic Glycogen stores are all characteristic of Von –Gierke’s disease.

Von –Gierke’s disease

Glycogen storage disease (GSD) type I, is also known as Von Gierke’s disease or hepatorenal Glycogenesis. Von Gierke  described the first patient with GSD type I in 1929.

Basic concept- Glycogen is a readily mobilised storage form of glucose. It is a very large, branched polymer of glucose residues that can be broken down to yield glucose molecules when energy is needed. Most of the glucose residues in glycogen are linked by α-1,4-glycosidic bonds. Branches at about every tenth residue are created by α-1, 6-glycosidic bonds. 

Figure-1- showing structure of Glycogen
Glycogen is not as reduced as fatty acids are and consequently not as energy rich.

Why do animals store any energy as glycogen? Why not convert all excess fuel into fatty acids? 

Glycogen is an important fuel reserve for several reasons-

1)  The controlled break down of glycogen and release of glucose increase the amount of glucose that is available between meals. Hence, glycogen serves as a buffer to maintain blood-glucose levels.

2)   Glycogen’s role in maintaining blood-glucose levels is especially important because glucose is virtually the only fuel used by the brain, except during prolonged starvation.

3)  Moreover, the glucose from glycogen is readily mobilised and is therefore a good source of energy for sudden,strenuous activity.

4)  Unlike fatty acids, the released glucose can provide energy in the absence of oxygen and can thus supply energy for anaerobic activity.

The two major sites of glycogen storage are the liver and skeletal muscle. The concentration of glycogen is higher in the liver than in muscle (10% versus 2% by weight), but more glycogen is stored in skeletal muscle overall because of its much greater mass. Glycogen is present in the cytosol in the form of granules ranging in-

 Figure-2- showing glycogen granules

-diameter from 10 to 40 nm .In the liver,glycogen synthesis and degradation are regulated to maintain blood-glucose levels as required to meet the needs of the organism as a whole. In contrast,in muscle, these processes are regulated to meet the energy needs of the muscle itself.

An Overview of Glycogen Metabolism

Glycogen degradation and synthesis are relatively simple biochemical processes.

Glycogen degradation consists of three steps:

 (1) The release of glucose 1-phosphate from glycogen,

(2) The remodelling of the glycogen substrate to permit further degradation, and

 (3) The conversion of glucose 1-phosphate into glucose 6-phosphate for further metabolism. (See Figure-2)

The glucose 6-phosphate derived from the breakdown of glycogen has three fates –

(a) It is the initial substrate for Glycolysis,

(b) It can be processed by the pentose phosphate pathway to yield NADPH and ribose derivatives; and

(c) It can be converted into free glucose for release into the bloodstream.

 This conversion takes place mainly in the liver and to a lesser extent in the intestines and kidneys.

 

 Figure -3- Showing glycogen degradation and the fate of glucose-6- phosphate.

Glycogen synthesis -requires an activated form of glucose, uridine diphosphate glucose (UDP-glucose), which is formed by the reaction of UTP and glucose 1-phosphate. UDP-glucose is added to the nonreducing end of glycogen molecules. Branching takes place after the addition of at least 12 glucose residues. As is the case for glycogen degradation, the glycogen molecule must be remodeled for continued synthesis.

The regulation of these processes is quite complex. Several enzymes taking part in glycogen metabolism allosterically respond to metabolites that signal the energy needs of the cell. These allosteric responses allow the adjustment of enzyme activity to meet the needs of the cell in which the enzymes are expressed. Glycogen metabolism is also regulated by hormonally stimulated cascades that lead to the reversible phosphorylation of enzymes, which alters their kinetic properties. Regulation by hormones allows glycogen metabolism to adjust to the needs of the entire organism. By both these mechanisms,glycogen degradation is integrated with glycogen synthesis. 

Figure-4 – showing an overview of glycogen metabolism

 Pathophysiology of Von –Gierke’s disease

Because of insufficient G6Pase activity,G6P cannot be converted into free glucose, but G6P is metabolised to lactic acid or incorporated into glycogen. In this way, large quantities of glycogen are formed and stored as molecules with normal structure in the cytoplasm of hepatocytes and renal and intestinal mucosa cells; therefore, enlarged liver and kidneys dominate the clinical presentation of the disease.

The chief biochemical alteration is hypoglycemia, while secondary abnormalities are hyperlactatemia, metabolic acidosis, hyperlipidemia, and hyperuricemia.

Hypoglycemia– The deficiency of G6Pase blocks the process of glycogen degradation and gluconeogenesis in the liver, preventing the production of free glucose molecules. As a consequence, patients with GSDtype I have fasting hypoglycemia. Hypoglycemia inhibits insulin secretion and stimulates glucagon and cortisol release.

Hyperlactatemia and acidosis– Undegraded G6P is metabolised to lactate, which is used in the brain as an alternative source of energy. The elevated blood lactate levels cause metabolic acidosis.

Hyperuricemia– Blood uric acid levels are raised because of the increased endogenous production and reduced urinary elimination caused by competition with the elevated concentrations of lactate, which should be excreted.

Hyperlipidemia– Elevated endogenous triglyceride synthesis and diminished lipolysis causes hyperlipidemia. Triglycerides increase the risk of fatty liver infiltration, which contributes to the enormous amount of liver enlargement. Despite significantly elevated serum triglyceride levels in patients, vascular lesions and atherosclerosis are rare complications.

Incidence

Patients with GSD type I account for 24.6%of all patients with GSD.

Inheritance

Type I glycogen storage disease is an autosomal recessive disorder .As with other genetically determined diseases,GSD type 1 develops during conception, yet the first signs of the disease may appear at birth or later.

 Clinical Manifestations

o  The earliest signs of the disease may develop shortly after birth and are caused by hypoglycemia and lactic acidosis.

o   Convulsions are a leading sign of disease.

o    Frequently,symptoms of moderate hypoglycemia, such as irritability,pallor, cyanosis, hypotonia, tremors, loss of consciousness, and apnoea, are present.

o   A leading sign of GSD type I is enlargement of the liver and kidneys. During the first weeks of life, the liver is normal size. It enlarges gradually thereafter, and in some patients, it even reaches the pubic symphysis. Enlargement of the abdomen due to hepatomegaly can be the first sign noted by the patient’s mother.

o   The patient’s face is characteristically reminiscent of a doll’s face (rounded cheeks due to fat deposition).

o   Mental development proceeds normally.

o   Growth is retarded and children affected with GSD type I never gain the height otherwise expected from the genetically determined potential of their families. The patient’s height is usually below the third percentile for their age. The onset of puberty is delayed.

o   Late complications of disease are renal function disturbance , renal stones, tubular defects, and hypertension, mainly in patients older than 20 years. Renal function deterioration progresses to terminal insufficiency,requiring dialysis and transplantation.

o   Skin and mucous membrane changes include the following:

  • Eruptive xanthomas develop on the extensor surfaces of the extremities.
  • Tophi or gouty arthritis may occur. Uric tophi often have the same distribution as xanthomas.
  • Many patients bleed easily, particularly from the nose. This tendency is a result of altered platelet function due to the platelets’ lower adhesiveness. Frequent and, occasionally, prolonged epistaxis may cause anaemia. At times, the bleeding may be so severe that blood transfusions are required.

 

Laboratory Investigations

GSD type I: Serum glucose and blood pH levels are frequently decreased, while the serum lactate, uric acid,triglyceride, and cholesterol levels are elevated. Urea and creatinine levels might be elevated when renal function is impaired. The following laboratory values should be obtained:

  • Serum glucose and electrolyte levels (Higher anion gap may suggest lactic acidosis.)
  • Serum lactate level
  • Blood pH
  • Serum uric acid level
  • Serum triglyceride and cholesterol levels
  • Gamma glutamyl transferase level (Liver dysfunction)
  • CBC and differential (eg, anaemia, leucopenia, neutropenia)
  • Coagulation- Bleeding and clotting time
  • Urinalysis for aminoaciduria, proteinuria, and microalbuminuria in older patients
  • Urinary excretion levels of uric acid and calcium
  • Serum alkaline phosphatase, calcium, phosphorus, urea, and creatinine levels.

Imaging Studies

  • In GSD type I, liver and kidney ultrasonography should be performed for follow-up of organomegaly.
  •  Abdominal CT scanning or MRI is advised whenever the lesions are large, poorly defined, or are growing rapidly.

Other Tests

  • Glucagon and epinephrine tests do not cause a rise in glucose levels, but plasma levels of lactic acid are raised.
  • Orally administered galactose and fructose (1.75 g/kg) do not increase glucose levels, but plasma lactic acid levels do increase.
  • Glucose tolerance test (1.75 g/kg PO) progressively lowers lactic acid levels over several hours after the administration of glucose.

Treatment

Most children with GSD type I are admitted to the hospital to make a final diagnosis, to manage hepatomegaly or hypoglycemia.

 Because no specific treatment is available,symptomatic therapy is very important.

 Diet 

 The primary goal of treatment is to correct hypoglycemia and maintain a normoglycemic state. The normoglycemic state can be achieved with overnight nasogastric infusion of glucose, parental nutrition, or per oral administration of raw corn starch. Glucose molecules are continuously released by hydrolysis of corn starch in the digestive tract over 4 hours following its intake. The intake of fructose and galactose should be restricted because it has been shown that they can not be converted to glucose but they do increase lactic acid production. Limited intake of lipids is advisable for the existing hyperlipidemia.

 Medication

·     No specific drug treatment is recommended for GSD type I. Appropriately treat concurrent infections with antibiotics.

·     Allopurinol (Zyloprim),a xanthine oxidase inhibitor, therapy can reduce uric acid levels in the bloodand prevent occurrence of gout and kidney stones in adult life.

·    Hyperlipidemia can be reduced by lipid-lowering drugs (eg, 3-hydroxy-3-methylglutaryl coenzyme A [HMG-CoA]reductase inhibitors, fibric acid derivatives).

·     In patients with renal lesions,microalbuminuria can be reduced with Angiotensin-converting enzyme (ACE)inhibitor therapy. In addition to their antihypertensive effects, ACE inhibitors are renoprotective and reduce albuminuria. Nephrocalcinosis and renal calculi can be prevented with citrate therapy.

·  Additionally, for patients with GSD type I,the future may bring Adeno-associated virus vector – mediated gene therapy,which may result in curative therapy,

Complications

  • Bacterial infections and cerebral oedema are caused by prolonged hypoglycemia and metabolic acidosis.
  • Long-term complications encompass growth retardation, hepatic adenomas with a high rate of malignant change, xanthomas, gout, and renal dysfunction. Long-term complications result from metabolic disturbances, mostly hypoglycemia.
  • Acute hypoglycemia may be fatal, and long-term complications include irreversible damage to the CNS.
  • Early death usually caused by acute metabolic complications (eg, hypoglycemia, acidosis) or  bleeding in the course of various surgical procedures

Prognosis

 The prognosis is better than in the past provided that all the available dietary and medical measures are implemented.

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Case Details

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

What is the most likely diagnosis?

What is the biochemical basis for all the presenting symptoms?
Which laboratory test would you request?
Case discussion The patient is most probably suffering from diabetic ketoacidosis. She is a known diabetic and the presenting symptoms like abdominal pain, vomiting, rapid breathing and distinctive smell of breath, all indicate associated ketoacidosis.
Basic concept Diabetic Ketoacidosis (DKA) is a state of inadequate insulin levels resulting in high blood sugar and accumulation of organic acids and ketones in the blood.  It is a potentially life-threatening complication in patients with diabetes mellitus. It happens predominantly in type 1 diabetes mellitus, but it can also occur in type 2 diabetes mellitus under certain circumstances.

Causes- DKA occurs most frequently in knownDiabetics. It may also be the first presentation in patients who had not been previously diagnosed as diabetics. There is often a particular underlying problem that has led to DKA episode. This may be-

1) Inter current illness such as Pneumonia,Influenza, Gastroenteritis, Urinary tract infection or pregnancy.

2) Inadequate Insulin administration may be due to defective insulin pen device or in young patient intentional missing of dose due to fear of weight gain.

3) Associated myocardial infarction, stroke or use of cocaine

4) Inadequate food intake– may be due to anorexia associated with infective process or due to eating disorder in children. 

Diabetic keto acidosis may occur in those previously known to have diabetes mellitus type 2 or in those who on further investigations turn out to have features of type 2 diabetes (e.g. obesity,strong family history); this is more common in African, African-American and Hispanic people. Their condition is then labelled “ketosis-prone type 2 diabetes”.

Pathophysiology

DKA results from relative or absolute insulin deficiency combined with counter regulatory hormone excess (Glucagon, Catecholamines, cortisol, and growth hormone). The decreased ratio of insulin to Glucagon promotes Gluconeogenesis,glycogenolysis, and Ketone body formation in the liver, as well as increases in substrate delivery from fat and muscle (free fatty acids, amino acids) to the liver. 

a) Cause of hyperglycemia Uncontrolled IDDM leads to increased hepatic glucose output.First, liver glycogen stores are mobilized then hepatic gluconeogenesis is used to produce glucose. Insulin deficiency also impairs non-hepatic tissue utilization of glucose. In particular in adipose tissue and skeletal muscle,insulin stimulates glucose uptake. This is accomplished by insulin-mediated movement of glucose transporter proteins to the plasma membrane of these tissues.

Reduced glucose uptake by peripheral tissues in turn leads to a reduced rate of glucose metabolism. In addition, the level of hepatic Glucokinase is regulated by insulin. Therefore, a reduced rate of glucose phosphorylation in hepatocytes leads to increased delivery to the blood. Other enzymes involved in anabolic metabolism of glucose are affected by insulin(primarily through covalent modifications). The combination of increased hepatic glucose production and reduced peripheral tissues metabolism leads to elevated plasma glucose levels.

b) Cause of kenosis 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 hepatocytes to synthesize triglycerides and storage of triglycerides in adipose tissue. In opposition to increased adipose storage of triglycerides is insulin-mediated inhibition of lipolysis. In uncontrolled IDDM 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. Normally, the levels of malonyl-CoA are high in the presence of insulin. These high levels of malonyl-CoA inhibit carnitine palmitoyl 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. However, in hepatocytes the majorityof the acetyl-CoA is not oxidized by the TCA cycle but is metabolized into the ketone bodies, Acetoacetate and β-hydroxybutyrate.  TCA cycle is in a state of suppression due to non availability of oxaloacetate which is channeled towards pathway of gluconeogenesis in the absence of Insulin. 

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.

c) Causes of Acidosis and hyperventilationThe ketone bodies, however, have a low pH and therefore turn the blood acidic(metabolic acidosis). The body initially buffers this with the bicarbonate buffering system, but this is quickly overwhelmed and other mechanisms to compensate for the acidosis, such as hyperventilation to lower the blood carbon dioxide levels. This hyperventilation, in its extreme form, may be observed as Kussmaul respiration. Ketones, too,participate in osmotic diuresis and lead to further electrolyte losses. As a result of the above mechanisms, the average adult DKA patient has a total body water shortage of about 6 liters (or 100 ml/kg), in addition to substantial shortages in sodium, potassium, chloride, phosphate, magnesium and calcium. Glucose levels usually exceed 13.8 mmol/l or 250 mg/dl. 

Increased lactic acid production also contributes to the acidosis. The increased free fatty acids increase triglyceride and VLDL production. VLDL clearance is also reduced because the activity of insulin-sensitive lipoprotein lipase in muscle and fat is decreased. Most commonly, DKA is precipitated by increased insulin requirements, as might occur during a concurrent illness. Occasionally, complete omission of insulin by the patient with type 1 DM precipitates DKA.

Clinical manifestations– The symptoms of an episode of diabetic ketoacidosis usually evolve over the period of about 24 hours. Predominant symptoms are nausea and vomiting, pronounced thirst,excessive urine production and abdominal pain that may be severe.

Hyperglycemia is always present .In severe DKA, breathing becomes labored and of a deep, gasping character (a state referred to as “Kussmaul respiration”). The abdomen may be tender to the point that an acute abdomen may be suspected, such as acute pancreatitis, appendicitis or gastrointestinal perforation.  

Coffee ground vomiting(vomiting of altered blood) occurs in a minority of patients; this tends to originate from erosions of the esophagus. In severe DKA, there may be confusion, lethargy, stupor or even coma(a marked decrease in the level of consciousness).

On physical examination -there is usually clinical evidence of dehydration, such as a dry mouth and decreased skin turgor. If the dehydration is profound enough to cause a decrease in the circulating blood volume, tachycardia (a fast heart rate) and low blood pressure may be observed. Often, a “ketotic”odor is present, which is often described as “fruity”. If Kussmaul respiration is present, this is reflected in an increased respiratory rate.

Small children with DKA are relatively prone to cerebral edema (swelling of the brain tissue), which may cause headache, coma, loss of the pupillary light reflex, and progress to death. It occurs in 0.7–1.0% of children with DKA, and has been described in young adults, but is  very rare in adults. It carries 20–50% mortality. 

 

Figure- showing causes and consequences of DKA

Diagnosis

Investigations-  Diabetic Ketoacidosis may be diagnosed when the combination of hyperglycemia (high blood sugars), ketones on urinalysis and acidosis are demonstrated.

Arterial blood gas measurement is usually performed to demonstrate the acidosis; this requires taking a blood sample from an artery.

In addition to the above, blood samples are usually taken to measure urea and creatinine (measures of kidney function, which may be impaired in DKA as a result of dehydration) and electrolytes.

Furthermore, markers of infection (complete blood count, C-reactive protein) and acute pancreatitis (amylase and lipase) may be measured.

Given the need to exclude infection, chest radiography and urinalysis are usually performed.If cerebral edema is suspected because of confusion, recurrent vomiting or other symptoms, computed tomography may be performed to assess its severity and to exclude other causes such as stroke.

Management

 The main aims in the treatment of diabetic ketoacidosis are replacing the lost fluids and electrolytes while suppressing the high blood sugars and ketone production with insulin.

a) Fluid replacement The amount of fluid depends on the estimated degree of dehydration. If dehydration is sosevere, rapid infusion of saline is recommended to restore circulating volume.

 b) Insulin is usually given continuously.

c) Potassium levels can fluctuate severely during the treatment of DKA, because insulin decreases potassium levels in the blood by redistributing it into cells. Serum potassium levels are initially often mildly raised even though total body potassium is depleted. Hypokalemia often follows treatment. This increases the risk of irregularities in the heart rate. Therefore, continuous observation of the heart rate is recommended, as well as repeated measurement of the potassium levels and addition of potassium to the intravenous fluids once levels fall below 5.3 mmol/l. If potassium levels fall below 3.3 mmol/l, insulin administration may need to be interrupted to allow correction of the hypokalemia.

d) Bicarbonate- Sodium bicarbonate solution is administered to rapidly improve the acid levels in the blood.

Cerebral edema- administration of fluids is slowed; intravenous Mannitol and hypertonic saline (3%) are used.

Prognosis

With appropriate therapy, the mortality of DKA is low (<5%) and is related more to the underlying or precipitating event, such as infection or myocardial infarction. The major non metabolic complication of DKA therapy is cerebral edema,which most often develops in children as DKA is resolving.

The etiology of and optimal therapy for cerebral edema are not well established, but over replacement of free water should be avoided. The other known complications of DKA therapy are, Hypoglycemia, hypokalemia and hypophosphatemia. Venous thrombosis, upper gastrointestinal bleeding, and acute respiratory distress syndrome occasionally complicate DKA.

Prevention of DKA

Following treatment, the physician and patient should review the sequence of events that led to DKA to prevent future recurrences. Foremost is patient education about the symptoms of DKA, its precipitating factors, and the management of diabetes during a concurrent illness.

During illness or when oral intake is compromised, patients should:

(1) frequently measure the capillary blood glucose;

(2) measure urinary ketones when the serum glucose > 16.5 mmol/L (300 mg/dL);

(3) drink fluids to maintain hydration;

(4) continue or increase insulin; and

(5) seek medical attention if dehydration, persistent vomiting, or uncontrolled hyperglycemia develop. Using these strategies, early DKA can be prevented or detected and treated appropriately on an outpatient basis.

DKA IN PREGNANCY-   DKA in pregnancy is of special concern. It tends to occur at lower plasma glucose levels and more rapidly than in non-pregnant patients and usually occurs in the second and third trimesters because of increasing insulin resistance. Fetal mortality rates have previously been reported as high as 30% rising to over 60% in DKA with coma. However with improvements in diabetic care the figure for fetal loss has been reported as low as 9% in some countries. Prevention, early recognition and aggressive management are vitally important to minimize fetal mortality.

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Case details

A pregnant woman who was extremely lactose intolerant asked her physician if she could still be able to breast feed her baby even though she could not drink milk or dairy products. What advice should be given?

The patient should be advised to breast feed the baby after delivery. Lactose is synthesized from UDP galactose and glucose; however galactose is not required in the diet for lactose synthesis, because galactose can be synthesized from glucose.

Lactose synthesis

Lactose is unique in the sense that it can only be synthesized in the mammary gland of the adult female for short period during lactation.Lactose synthase present in the endoplasmic reticulum of the lactating mammary gland, catalyzes the last step in the lactose biosynthesis, the transfer of galactose from UDP galactose to glucose.

Figure-showing synthesis of Lactose from UDP galactose

Lactose synthase attaches the anomeric carbon of galactose to C4 alcohol group of
glucose to form a glycosidic bond. Lactose synthase is composed of a galactosyl transferase and α lactalbumin which is a regulatory subunit. Lactose synthase is activated by Prolactin hormone. In the non lactating mammary glands, this enzyme is inactive, so lactose is not synthesized and UDP galactose is alternatively used for the formation of glycolipids or glycoproteins.

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Case Details

A 54-year-old army officer presented to a clinic for follow-up of his diabetes. He seemed very particular about his health. He had diabetes since the age of 12 and had been on insulin therapy. He had been regularly monitoring his blood glucose as advised by the physician.

He reported feeling very restless, diaphoretic and felt hunger cramps at 2 AM with the blood sugar in the range of 40 mg/dL. He, however, noted that his morning fasting blood sugar was high without taking any carbohydrates. His physician described the morning high sugars as a result of biochemical processes in response to the night-time hypoglycemia.

What are the biochemical processes that govern the response to the night-time hypoglycemia?

Case Discussion

The night-time hypoglycemia stimulates the counter-regulatory hormones to raise the blood glucose levels. These include epinephrine, glucagon, cortisol, and growth hormone, which raise the glucose level by morning.

The patient in the given case has the classic manifestations of Somogyi effect, also known as “rebound hyperglycemia” named after the physician who first described it. The Somogyi effect is a pattern of undetected hypoglycemia (low blood glucose values of less than 70) followed by hyperglycemia (high blood glucose levels of more than 200). Typically, this happens in the middle of the night, but can also occur when too much insulin is circulating in the system.

Over dosage of insulin seems to be the cause of Somogyi effect. The diagnosis is established by measuring a 2 AM blood glucose level, and  upon confirmation, the bedtime insulin dose is decreased.

The danger is that if night-time blood glucose levels are not measured, the physician may interpret the patient as having hyperglycemia and require even higher doses of insulin. This would be exactly the wrong treatment, since it would worsen the situation by causing hypoglycemia leading to counter-regulatory hormone reaction, and a very high sugar level in the morning.

Another similar phenomenon is also observed called Dawn phenomenon, named after the time of day it occurs. The “dawn effect,” also called the “dawn phenomenon,” is the term used to describe an abnormal early morning increase in blood sugar (glucose) — usually between 2 a.m. and 8 a.m. — in people with diabetes.

Some researchers believe it’s due to the natural overnight release of hormones —including growth hormones, cortisol, glucagon and epinephrine — that increase insulin resistance.

High morning blood sugar may also have other causes. Insufficient insulin the night before, incorrect medication dosages or eating carbohydrate snacks at bed time cause blood sugar to get elevated in the morning. When necessary, checking blood sugar once during the night — around 2 a.m. or 3 a.m. — can help to differentiate if there is dawn phenomenon or if there’s another reason for an elevated morning blood sugar level.

 

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Case details

A 23 –year- old female was brought for consultation by her mother who was troubled by her daughter’s continuous fatigue, dizziness and loss of weight. The patient was 6 feet 2 inches tall and weighed 100 pounds.

Laboratory results revealed – Blood glucose 50 mg% and elevated ketones.

Further questioning revealed that the young woman had been virtually fasting for 4 months hoping to obtain a ‘skinny figure’ as a prelude to a career in modeling.

What is the problem with this female?

What is the cause of hypoglycemia?

Case discussion

The patient shows many signs of Anorexia Nervosa (AN), which is an eating disorder. The etiology of AN is unknown but appears to involve a combination of psychological, biologic, and cultural risk factors. The condition is characterized by aversion to food that leads to a state of fasting and emaciation. Patients often have a distorted image of their own body weight or shape and are unconcerned by the serious health consequences of their low weight. As weight loss progresses, the fear of gaining weight grows; dieting becomes stricter; and psychological, behavioral, and medical aberrations increase.

After several months of near starvation, the blood glucose in these patients is maintained by gluconeogenesis, primarily from amino acids mobilized from tissue proteins. The patients develop potential vitamin deficiencies. Liver glycogen is exhausted in the first day of fasting. Gluconeogenesis depletes the supply of oxaloacetate, which is essential for the entry of acetyl CoA into the citric acid cycle. Consequently, the liver produces large quantities of ketone bodies, which are released into the blood. After several weeks of starvation, ketone bodies become the major fuel of the brain. The impaired conversion of amino acids in to glucose is responsible for producing hypoglycemia in these patients. A person’s survival time is mainly determined by the size of the triacylglycerol depot.

The diagnosis of AN is based on the presence of characteristic behavioral, psychological, and physical attributes. Psychotherapy, medication, or hospitalization are required to treat these patients.

 

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Case details

A 65-year-old man was admitted to the emergency department in an unconscious state. Apparently he had become increasingly depressed after death of his younger son two months ago. Previously before his death he had been a moderate drinker, but consumption of alcohol had increased markedly over the last few weeks. He had also been eating poorly,

His elder son had dropped around to see him on Sunday morning and found him unconscious in the living room couch with two empty bottles of whisky. Three  more bottles were also found on the living room table.

On examination he could not be roused, his breathing was deep and noisy,

Alcohol could be smelt in his breath, and his temp was 36.6° C.

Lab findings:

Blood alcohol 550 mg/dl

Blood glucose 50 mg/dl

Blood lactate 8 mmol/L

pH 7.21

 What is the biochemical basis for all the laboratory findings in this patient?

Case Discussion

This is a case of Alcohol (Blood alcohol-550mg/dL) induced hypoglycemia (Low glucose-50 mg/dl) and metabolic acidosis. Metabolic acidosis as apparent from low p H (7.21), is due to underlying lactic acidosis (Blood Lactate-8mmol/L).

Alcohol-related hypoglycemia is due to hepatic glycogen depletion combined with alcohol-mediated inhibition of Gluconeogenesis. It is very common in malnourished alcohol abusers but can occur in anyone who is unable to ingest food after an acute alcoholic episode followed by gastritis and vomiting.

The primary pathway for alcohol metabolism involves alcohol dehydrogenase (ADH), a cytosolic enzyme that catalyzes the conversion of alcohol to acetaldehyde. This enzyme is located mainly in the liver, but small amounts are found in other organs such as the brain and stomach.

 During conversion of ethanol by ADH to acetaldehyde, hydrogen ion is transferred from alcohol to the cofactor nicotinamide adenine dinucleotide (NAD+) to form NADH (Figure- Step-1).

 

Much of the acetaldehyde formed from alcohol is oxidized in the liver in a reaction catalyzed by mitochondrial NAD-dependent aldehyde dehydrogenase (ALDH) (Figure-Step-2) .

 The product of this reaction is acetate, which can be further metabolised to CO2 and water, or used to form acetyl-CoA. As a net result, alcohol oxidation generates an excess of reducing equivalents in the liver, chiefly as NADH. The excess NADH production appears to contribute to the metabolic disorders that accompany chronic alcoholism.

 1)   The NADH produced in the cytosol by ADH must be reduced back to NAD+ via either the malate-aspartate shuttle or the glycerol-phosphate shuttle. Thus, the ability of an individual to metabolize ethanol is dependent upon the capacity of hepatocytes to carry out either of these 2 shuttles, which in turn is affected by the rate of the TCA cycle in the mitochondria whose rate of function is being impacted by the NADH produced by the ALDH reaction.

 2)   The reduction in NAD+ impairs the flux of glucose through glycolysis at the glyceraldehyde-3-phosphate dehydrogenase reaction, thereby limiting energy production.

 3)   Additionally, there is an increased rate of hepatic lactate production due to the effect of increased NADH on direction of the hepatic lactate dehydrogenase (LDH) reaction. This reversal of the LDH reaction in hepatocytes diverts Pyruvate from Gluconeogenesis leading to a reduction in the capacity of the liver to deliver glucose to the blood.

 4)   Similar to lactate formation, Malate is also produced from Oxaloacetate. Deficiency of Oxaloacetate negatively affects Gluconeogenesis as well as the functioning of TCA cycle.

 5)   In addition to the negative effects of the altered NADH/NAD+ ratio on hepatic Gluconeogenesis, fatty acid oxidation is also reduced as this process requires NAD+ as a co factor.

 6)   In fact the opposite is true, fatty acid synthesis is increased and there is an increase in triglyceride production by the liver. In the mitochondria, the production of acetate from acetaldehyde leads to increased levels of acetyl-CoA. Since the increased generation of NADH also reduces the activity of the TCA cycle, the acetyl-Co A is diverted to fatty acid synthesis.

 7)    The reduction in cytosolic NAD+ leads to reduced activity of glycerol-3-phosphate dehydrogenase (in the glycerol 3-phosphate to DHAP direction) resulting in increased levels of glycerol 3-phosphate which is the backbone for the synthesis of the triglycerides. Both of these two events lead to fatty acid deposition in the liver leading to fatty liver syndrome.

 8)   Increased [lactate]/[Pyruvate] ratio, results in hyperlacticacidemia. Lactate accumulation causes lactic acidosis (Metabolic acidosis).

 9)    Lactate competes with uric acid for excretion, decreasing its excretion and thus aggravating gout. Gout is a common finding in chronic alcoholics.

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