12/3/2014 2001 Biochemistry Midterm

BIOCHEMISTRY MIDTERM 2001

A mother brought her two children to the family doctor. The older girl had had unexplained bouts of
serious anemia on several occasions. In addition, the older girl's sclera always looked yellowish, and her
hemoglobin was at the lower end of normal. The younger girl had not had any problems

Since the family was of Mediterranean extraction where G6PD (glucose-6-phosphate dehydrogenase)
deficiency is a relatively frequent defect the physician sent out blood samples of both children to a
laboratory to be checked for abnormalities of G6PD.

1. The enzyme of the younger child would be normal since she never had had an attack of
anemia.

The lab report came back with the following kinetic parameters for the enzymes of the two
children.

Kinetic parameters normal Older child Younger child

Km NADP+, µM 8 160 8
Km G6P, µM 50 50 50
Vmax, M/s 10 2.5 10
Ki NADPH, µM 9 9 90

2. The concentration of NADP+ in normal red cells is very low because the reduction to
NADPH is virtually irreversible.

3. G6PD was mutated in both children.

4. The anemia of the older child was explained by the decrease in Vmax since the pentose shunt
in red cells normally operates at Vmax.

5. The attacks of anemia in the older child were the result of a decrease in red cell ATP due to a
decrease in glycolytic flux that was caused by an abnormally low entry rate of F6P and GAP from
the pentose shunt into the glycolytic path. .

6. If the physician had measured erythropoietin in the older child's blood, she would have
found it to be higher than normal.

7. In the younger child, the NADP+ concentration necessary for half maximal rate of G6PD in
her red cells was much lower than in the normal.

8. If both children were exposed to an oxidative challenge, the hemolytic crisis would have been
worse for the younger child than the older child because her Ki for NADPH had been greatly
altered.

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Legend: This is a substrate saturation curve for normal ß-cell glucokinase.

Glucokinase has two Km values, one for glucose and another for ATP. The Km for ATP is 0.25
mM, and the intracellular ATP concentration is 5 mM, i.e. it is 20 times higher than the KmATP.
A patient has a mutation of the glucokinase that raises its Km for ATP to 50 mM, without
changing the kcat or the Km for glucose.

9. The mutation lowers the saturation of glucokinase with ATP from about 95% to about 10%.

10. Since the kcat is not changed the rate of G6P production at all glucose concentrations is the
same as in the normal.

11. The mutation decreases Vmax in the patient's ß-cells about 9fold so that the rate never
reaches set-point velocity at any glucose concentration.

12. The mutation has no effect on insulin secretion because the Km for glucose has not been
changed.

13. The mutation completely eliminates all control of the blood glucose level by the ß-cells.

14. To control blood glucose, the patient must be treated with insulin injections.

An 18 year old male who had complained for 2 years of brownish discoloration of the urine and easy
fatigue after and during hard exercise was referred to the hospital for further evaluation. Serum muscle
creatine kinase was very high. The patient was given a standard ischemic exercise test of 60 sec that
consisted of hand exercises. After 25 sec the patient started to complain of serious fatigue. After 60 s the
cuff cutting off the blood supply to the hand was removed, and venous blood was collected as shown in
the figure below.

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Legend: The closed circles indicate the highest and lowest lactate values found in several healthy
control subjects.

15. The high rate of lactate formation in the controls indicates that during exercise all muscles
always obtain all their ATP from glycolysis.

16. The results from the ischemic exercise test rule out a defect in glycogen degradation in the
patient’s muscles.

17. A high concentration of muscle creatine kinase in the patient’s blood indicates damage to the
muscle fibers.

Among the serum enzymes, the concentration of the muscle isoform of LDH was abnormally low
relative to the concentration of the muscle isoform of creatine kinase (the LDH/CK ratio). The
physicians discovered that the reason was an unusually low LDH content in the patient’s muscles.

18. The patient's attacks of muscle breakdown and muscle fatigue cannot be related to the LDH
deficiency of the muscles since ATP formation is completed at the PEP --> pyruvate step.

19. To the contrary, the large release of energy associated with NADH oxidation by LDH is
essential for ATP formation.

20. Glycolytic flux in this patient's glycolytic muscle fibers (very low in mitochondria) is limited
by NAD recycling rather than by the ADP supply.

21. In general, a severe inhibition of glycolytic flux can limit flux through the citric acid-cycle-
ox.phos.
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The patient was a 22 yr old man with lifelong exercise intolerance marked by premature fatigue.
He recalled that he never could walk as far or as long as other children. His calf muscles (largely
mitochondria rich muscle fibers) were especially affected. In addition to pain and fatigue, he had
occasional attacks of myoglobinuria. The patient was tested on the bicycle ergometer: his
maximal work capacity was about 1/6 of a normal man of his age as shown in Table I

Table I

Patient Control

Rest exercise rest exercise

Work capacity: 0.5 3.0

Watt/kg tissue

O2 consumption 5 12 5 40
ml/min x kg tissue

ml O2/min/watt 15 15
(rate of work performed)

Venous lactate 3 10 1 5
mM

Venous oxygen saturation 80 75 80 25

22. In general, the rate of ATP rephosphorylation in a muscle is not related to the rate of work
performed (watt).

23. In the control, the increase in oxygen consumption going from rest to exercise is caused by
the increase in ATP hydrolysis associated with exercise.

24. The patient performed much less exercise because he had less oxygen in his blood.

25. The ratio of the rate of O2 consumption and the rate of work performed (watt) , i.e., ml
O2/min/watt would have been abnormally high if the patient had had a proton leak.

26. The ratio of the rate of O2 consumption and the rate of work performed (watt) , i.e., ml
O2/min/watt would have been lower than normal if the patient's muscles had had an abnormally
high proportion of glycolytic fibers.

27. The observation that the rate of oxygen consumption /watt was the same for the control and
the patient indicates that his mitochondria had a P/O ratio of about 3.

28. It is reasonable to assume that the patient hydrolyzed less ATP per kg muscle than the
control.

29. The exercise-induced increase in the venous lactate level of the control is largely derived from
the glycolytic fibers of the muscles (low in mitochondria).

30. All of the patient's abnormalities listed so far would be explained if the patient's
mitochondria had a defect that prevented them from rephosphorylating all of the ADP generated by
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exercise.

To investigate the cause of the exercise intolerance, mitochondria were isolated from a biopsy
taken from the patient's calf muscles. The oxygen consumption in the presence and absence of
ADP was measured, using two different substrates (Table II).

Table II

Oxygen consumption ( nano atoms / min / mg protein)

Substrate Pyruvate (+malate) malate

Control - ADP 40 40

Control + ADP 190 190

Patient - ADP 40 40

Patient + ADP 60 190

31. In general, oxygen consumption in the absence of ADP is the consequence of a small degree
of proton leak that is always present.

32. In the control, the increase in the rate of oxygen consumption after ADP addition indicates
the tight coupling of electron transport and ADP phosphorylation.

33. The data in Table II indicate that the patient's problems are caused by an abnormally large
proton leak.

34. All of the data presented here could be explained by a bottleneck in the patient’s citric acid
cycle.

Next, the calf muscle was biopsied again, and the Vmax of several enzymes was measured in the
homogenized mitochondria. The results are shown in Table III.
Table III

Enzyme Activity Control Patient

µmoles/min/mg protein
Citrate synthase 10.6 14.8

Succinate dehydrogenase 4.2 1

Fumarase 8.9 9.1

Malate dehydrogenase 170 241

35. Flux through the citric acid cycle should have been faster in the patient than in the control
because the Vmax for citrate synthase was higher than that in the control.

During thiamine deficiency

36. The activities of PDH and a‑ ketoglutarate dehydrogenase are deficient.

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37. During thiamine deficiency heart function is better during a12h fast than after a glucose
infusion, which may cause acute cardiac failure.

38. Similarly, brain function also is much better during a 12h fast than after a carbohydrate meal.

39. Glycolytic flux in the nervous system will be unusually high during fasting and feeding.

40. The conversion of pyruvate to glucose is blocked.

41. The rate of lactic acid production (resulting in lactic acidosis) is lower at a low I/G than at a
high I/G in most, but not all, tissues,

The patient, a 14 year old boy of normal growth and superior intelligence has been cyanotic
(desoxygenated Hb is blue compared to the pink color of Hb-O2) since birth (blue as in
asphyxiation). Nevertheless, he had never been short of breath, and participated in all sports
activities at his school. However, after severe exercise he sometimes felt weak and had to take a
break. Pulmonary function tests were normal and did not explain his cyanosis. His heart and
circulation also were normal. A test of the hemoglobin in his red cells was made as shown in
Figure 1.

Legend: The oxygen saturation of his hemoglobin was compared with that of normal Hb. A
suspension of red cells of each (control and patient) was exposed to increasing oxygen under
identical conditions (e.g. pH, CO2, salts).

42. The patient's Hb binds much less oxygen than the normal Hb.

43. The oxygen saturation curve is consistent with a severe hemoglobin deficiency (anemia).

44. The oxygen saturation curve suggests that oxygen has been displaced by CO.

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45. Because of the low oxygen saturation of the patient's hemoglobin in the lung, his oxygen
delivery to the tissues is greatly impaired.

46. The patient would be expected to compensate by an increase in the red cell number.

47. Having the patient inhale 100% oxygen instead of air (20% oxygen) would increase his
maximally possible oxygen delivery way above the maximally possible oxygen delivery of normal
hemoglobin.

48. The patient's ability to carry out excessive exercise is not impaired.

A 7 year old boy was admitted to the hospital because he had a seizure. On admittance his blood sugar
was below 1.5 mM. He was stabilized with a glucose infusion and then watched in the hospital on a
normal diet. Since he remained stable he was sent home. When he had another seizure several months
later, he was admitted for a metabolic study. Some of the data are shown in the Figure below.

Legend: The patient was subjected to a 24h fast and blood glucose, lactate and ketones were
measured.

These data were given to a group of first year students for interpretation. Here is an excerpt from their
discussion.

49. The high rate of fatty acid oxidation, indicated by the increase in blood ketone level, is
consistent with a hyperinsulinemia as a cause of the hypoglycemia.

50. The low blood lactate after the 24 hour fast is compatible with a low rate of alanine release
from muscle.

51.              A cortisol deficiency would have resulted in the backup of lactate.

52. The boy would have been much better off if the increase in the rate of fatty acid oxidation
had not occurred.

53. A fructose infusion at 12 pm (18h fast) would have increased the lactate level in the blood
(The blood fructose level has no effect on insulin release).

54. The low lactate level during the 24hfast allows one to conclude that fructose feeding at 12 pm
would have raised the blood glucose to normal.

55. The data in figure 1 lead one to conclude that the boy needed to be maintained on a 5h CHO
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feeding schedule.

56. The glucose concentration in the brain cells is the same as that in the blood.

57. Because the brain is so dependent on glucose the diffusion rate of glucose through the brain
capillaries is much faster than glucose diffusion through the capillaries in all other organs and
tissues.

58. Patients are confused when blood glucose levels are below 2 mM because glycolytic flux in
some of the brain cells starts to be limited by the glucose supply rather than by the ADP supply.

59. A severe decrease in the rate of glycolytic flux makes no difference to the ATP supply for the
brain cells because their ATP is rephosphorylated by the citric acid cycle-ox.phos.

60. Patients are confused when blood glucose levels are below 2 mM because the rate of ion
pumping is falling below normal.

61. Patients with an insulinoma are confused when the high insulin levels prevent the brain
phosphorylase from releasing glucose from brain glycogen.
Question 4.

62. After glucose ingestion some insulin is released before the blood glucose level has increased.

63. Any process that releases calcium into the cytosol of the ß-cell stimulates the release of
insulin because insulin release depends on the exocytosis of insulin-containing vesicles.

64. Insulin release after the uptake of amino acids from the gut promotes protein synthesis in
muscle.

65. The amount of insulin synthesized per day by the ß-cells is much greater in a patient with
insulin resistance than in a healthy control.

A 7 year old girl was admitted to the hospital for a metabolic study because of a history of early
morning convulsions. She was small, below the 3rd percentile both in height and weight. On the
morning after admission she was drowsy and her blood glucose was 1.1 mM. A 24 h metabolic
profile was made: plasma glucose and lactate were measured, as shown in the figure below.

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A student and the intern discussed the interpretation of the results of the 24 metabolic study.

66. The blood chemistry between 12pm and 12 am was the same as that expected for an
untreated diabetic.

67. The overshoot in blood glucose curve is compatible with deficient glucose incorporation into
muscle glycogen.

68. The high level of lactate could have been caused by an increase in glycolytic flux of resting
muscle caused by the increase in blood glucose.

69. The fasting hypoglycemia could have been the result of excessive glucose uptake into the
resting muscle due to high insulin.

70, the hypoglycemia beginning 6h after the last meal could be caused by a defect in the
conversion of liver glycogen to blood glucose.

The intern ordered a galactose-feeding test after an over night fast. This brought the blood
glucose back up to 6 mM.

71. This outcome is compatible with G6Pase deficiency since this is a defect between pyruvate
and glucose and galactose comes in at G1P.

72. A debrancher deficiency would have presented with the metabolic changes shown in the
figure.

73. The metabolic study alone was insufficient for a diagnosis of debrancher deficiency.

The attending ordered the counter regulatory hormones measured after the 12h fast. They were
all much lower than they had expected for the extent of hypoglycemia.

74. The patient must have an insulinoma.

75. Frequent attacks of hypoglycemia shift the release of the counterregulatory hormones to
much lower blood glucose values.

76. The patient had a defect in liver glycogen metabolism.

A 5-yr old girl in a coma was rushed to the hospital some time in the morning. The day before,
she had felt quite sick with a stomach virus, had vomited several times and eaten very little. The
child had been breast fed for 7 months and she had thrived. Over the later years, she had been
dizzy in the morning on several occasions but recovered after breakfast.

Table I shows a few blood chemistry data obtained on admission.

Table I

Patient Control

Glucose mM 1.8 You know

Pyruvate mM 1.8 0.1

CO2 + HCO3- mM 5 25

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The data in Table I indicate that

77. The child’s pH must be below 7.3; i.e. the child must be acidotic.

78. The data allow you to conclude that the blood lactate was most likely about 18 mM.

79. The child most likely had a defect in glycogen degradation.

After the child had recovered, she was subjected to a metabolic study, starting with a 21 h fast.
Glucose and lactate levels were followed over time. The results are shown in Figure 1

Legend Figure 1: the solid curve represents the glucose measurements and the lactate data by the
dashed curve

80. The decrease in glucose after a 14h fast is compatible with a block in gluconeogenesis
between pyruvate and glucose.

81. The fall in blood glucose after a 14h fast is characteristic of G6Pase deficiency.

82. The lactate originated from glycogen in resting muscle in response to the very low
insulin/glucagon ratio.

83. The carbons of the lactate produced during the metabolic study are derived from alanine
released by the muscles in response to the high cortisol and low insulin.

Next, the child was given a glucagon test. The results are shown in Figure 2

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Legend Figure 2: Glucagon was injected at time zero.

84. In general, liver glycogen is released after injection of a large amount of glucagon because the
glucagon injection lowers I/G sufficiently to maximally inhibit liver glycogen synthase and
maximally activate liver phosphorylase.

85. The normal increase in blood glucose after a 6 hr fast is compatible with a problem with
G6Pase.

Next, alanine was infused into the patient when he was hypoglycemic and the blood levels of glucose,
lactate and alanine were measured.

86. The alanine level decreased when alanine was excreted in the urine.

87. Alanine did not go to glucose because the I/G was too low.

88. The lactate level went up because alanine allosterically activates PFK.

89 the outcome of the alanine test is compatible with both G6Pase deficiency and FBPase
deficiency.

90. All findings described here would be explained by a FBPase deficiency.

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