AN APPLE A DAY KEEPS THE DOCTOR IN PAY

1. AN APPLE A DAY KEEPS THE DOCTOR IN PAY http://www.toothpastefordinner.com/ BCHM2972 Lecture 10 2006

2. Presentation and history: • A 10-year old girl, Jane, has a long history of problems after eating fruit or any foods containing sugar. • The symptoms include vomiting, stomach cramps, trembling, sweating, dizziness, ~ convulsions.  • 'sickly' since weaned from breast milk to infant formula and solids.  • OK foods?  trial and error. • glucose OK but she does not enjoy the taste. • cow's milk OK • physical development • Difficult to maintain sugar free diet. • teeth show no caries!

3. Jane has presented at hospital tonight with violent convulsions which were preceded by stomach cramps and sweating. • After soft drink and lollies at a friend's slumber party (peer pressure!) • Refer to the handout of Test results

4. What is the likely cause of the sweats, convulsions, etc? A Hypoglycemia B Hyperglycemia C Hypoinsulinemia D Hyperfructosemia

5. glucose sucrose galactose fructose lactose Which ‘sugars’ is Jane reacting to?

6. glucose sucrose galactose fructose lactose We know foods with glucose OK cows milk OK FRUIT a problem lollies and soft drinks too Which ‘sugars’ is Jane reacting to?    monosaccharides sucrose fructose + glucose invertase in small intestine absorbed to blood

7. no fructose in breast milk! Aldolase B not expressed in infants long history always ‘sickly since weaning’ Why not since birth? so…a problem with fructoseinborn error of metabolism?

8. which do you expect to be abnormal? fructose tolerance test:infuse 200mg/kg fructose intravenously measure blood levels of: fructose glucose phosphorus magnesium uric acid confirmed blood test levels are in response to fructose

9. What the…? • Jane has a problem eating fructose, but fructose clearance is normal !! • Why is glucose  ( symptoms) ? • ……and why  P and  Mg? • and uric acid and bilirubin?? • don’t these mean liver damage?

10. It appears fructose is very efficiently taken up and trapped in tissues • it's the metabolism of the fructose in the tissues that's the problem So…..let’s review “sugar” metabolism

11. nucleophile (donates e-) 1st step in glycolysis Breakage of the Phosphoanhydride Bond in ATP

12. Glucose ATP P PHOSPHORYLATION hexokinase isomerase ISOMERISATION Fructose 6 P 6 glycolysis ATP phospho fructo kinase PHOSPHORYLATION Aldolase A ‘CLEAVAGE’ ~30 ATP per glucose P P DHAP GA 3 P CITRIC ACID CYCLE electron transport and OxPhos Pyr deHydr ATP pyruvate

13. Glucose Fructose ATP P hexokinase ATP hexokinase isomerase  affinity for fructose Fructose 6 P 6 glycolysis phospho fructo kinase in muscle, fat cells ATP Aldolase A P P DHAP GA 3 P CITRIC ACID CYCLE electron transport and OxPhos ATP pyruvate

14. Glucose Fructose ATP ATP hexokinase fructokinase in liverhexokinase hasaffinity for glucose but affinity for fructose. lots of glucose in liver so instead of hexokinase, fructokinase adds P to trap fructose in cells Fructose 1 P Fructose 6 P 1 6 glycolysis ATP Aldolase B ATP P Glyceraldehyde (GA) P P DHAP GA 3 P DHAP CITRIC ACID CYCLE electron transport and OxPhos ATP pyruvate

15. What is the result of the first step in fructose metabolism? • phosphorylation of fructose • ATP used up to ph’late fructose • cellular energy is reduced • phosphate ‘trapped’ in fructose 1 P • all of the above

16. What is the result of a mutation in fructokinase? • can’t ph’late fructose  fructose- 1P • reduced clearance of fructose from blood • blood fructose remains high • ATP not used to p’late fructose • all of the above

17. 12 10 8 fructose (mg/dL) 6 4 2 0 -2 0 30 60 90 120 time (min) Can Jane’s liver trap fructose in the tissue (clearing it from blood)? • Yes • No Does Jane have a mutation in fructokinase? • Yes • No Her blood fructose does not rise rapidly after the fructose load because fructokinase is very good a mopping up and trapping fructose in the liver. And/or hexokinase in other tissues is very good at mopping up fructose into F6P

18. Which enzyme catalyses the second step of fructose catabolism? • hexokinase • fructokinase • phosphofructokinase • aldolase A • aldolase B

19. What is the result of a mutation in aldolase B? • fructose -1P builds up • ph’lation of F-1P uses up ATP • cellular energy is reduced • phosphate ‘trapped’ in fructose- 1 P • all of the above

20. Fructose Glucose ATP fructokinase ATP ATP 1 Fructose 1 P glycolysis Aldolase B 30 ATP pyruvate

21. Why the  Pblood? How do we explain Jane’s results by a mutation in aldolase B? • P trapped in fructose 1-P • P trapped in fructose 6-P • P trapped in fructose 1, 6 -biP • P trapped in glyceraldehyde 3P • all of the above

22. How do we explain Jane’s results by a mutation in aldolase B? Why the  Pblood? • P trapped in fructose 1-P • P trapped in fructose 6-P • P trapped in fructose 1, 6 -biP • P trapped in glyceraldehyde 3P • all of the above

23. What are the consequences of  Pblood? • ADP + P  ATP low cellular energy (ATP) cell damage /death  release of liver enzymes and bilirubin

24. 0.5 0.4 0.3 magnesium (mg/dL) 0.2 0.1 0 0 30 60 90 120 time (min) Why the high Mg? • ATP in complex with Mg2+ • ATP  Mg2+ released to blood

25. ATP, Mg2+, and +ve residues at an enzyme active site Since all the negative charges in ATP are neutralized, ATP is readily approached by nucleophiles

26. substrate level phosphorylation to compensate for ATP Glucose ATP P hexokinase ATP ADP Fructose isomerase ADP AMP Fructose 6 P 6 + glycolysis phospho fructo kinase ATP  PFK rate gylcolysis (important regulatory point) Aldolase A P P DHAP GA 3 P CITRIC ACID CYCLE electron transport and OxPhos ATP pyruvate

27. CITRIC ACID CYCLE ATP electron transport and OxPhos normally…… NAD+ PFK PDH AcCoA glucose pyruvate glycolysis NADH

28. CITRIC ACID CYCLE ATP electron transport and OxPhos NAD+ AMP glycolysis + PFK PDH AcCoA glucose pyruvate NADH In Jane’s liver, pyruvate is produced too fast to enter mitochondria for TCA (PDH is like the ‘plughole’) NAD not regenerated

29. cell must regenerate NAD+ to keep glycolysis running desperate attempt to make ATP by glycolysis (2 per glucose) NAD+ glycolysis lactate glucose pyruvate ATP NADH

30. What processes normally lead to increased blood lactate? A Decreased liver gluconeogenesis B Increased liver glycolysis (This happens in Jane via stimulation of PFK by AMP) C Increased muscle glycolysis D Decreased PDH activity (would  entry of substrates to Citric Acid Cycle) E All of the above all increase pyrlactate to regenerate NAD

31. Fructose glycogen phosphorylase glycogenolysis ATP fructokinase Fructose 1 P P Glucose fructose 1,6 bisphosphatase Aldolase B gluconeogenesis Why is glucose sooooo low?in addition to  glycolysis…. Fructose 1,6 BP

32. What is  [uric acid*] blood indicative of? A  cellular 'energy charge' B  rate of deamination of adenosine C  in ATP D  in AMP E All of the above *not to be confused with urea! (product of amino acid catabolism)

33. H H H P P uric acid is a product of purine base degradation deamination AMP inosine MP uric acid

34. What problems can fructose cause for ‘normal’ people? • does NOT stimulate insulin or leptin • regulators of energy intake and body adiposity • bypasses phosphofructokinase • determines the rate of glycolysis (pyruvate) • So…..  fructose  acetyl CoA Lipogenesis liver TG insulin sensitivity  VLDL

35. Hepatic fructose metabolism: A highly lipogenic pathway. http://www.nutritionandmetabolism.com/content/figures/1743-7075-2-5-2-l.jpg

36. In Summary… You should make a summary diagram yourselves! Show main control points, interconnections, and show how the non-functioning enzyme lab results