Pentose Phosphate Pathway

Question 1

NADPH

Answer 1

1. Similar to NADH but this one has additional phosphate group
2. NADPH and NADH have same redox potential, but are metabolically disinct. NADPH utilizes its reductive potential for the synthesis of fatty acids and as an anti-oxidant.
3. Hepatocytes contain 100 times more NAD+ than NADH (oxidative role), while they contain 100 times more NADPH than NADP+ (reductive role)
4. This reflects the different functions of these coenzymes, where NADH is used up more actively as a source of energy for endergonic reactions.

Question 2

Free radicals

Answer 2

1. A free radical is defined as a molecule that contains a single unpaired electron in an orbital. Radicals are very reactive.
2. Radicals can initiate chain reactions of “electron grabbing.” These oxidations of neighboring molecules allow the radicals to complete their own orbitals.
3. Oxygen free radicals are known as ROS (Reactive Oxygen Species)
4. ROS are extremely short-lived and highly reactive. ROS cause the formation of free radical chain reactions.
5. The most physiologically significant ROS are the following:
   
   1. Superoxide (O₂^-)
   2. Peroxide (H₂O₂)
   3. Hydroxyl Radiol (OH^-); most devastating
6. The predominant source of ROS is the electron transport chain (ETC)
   
   1. The presence of high numbers of ROS is a factor in the high mutation rate of mitochondrial DNA, thus mitochondria goes through mutation 10x more than regular DNAs
7. Other sources include:
   
   1. The peroxisomal formation of hydrogen peroxide, which is converted to a ROS
   2. The microsomal and mitochondrial cytochrome P450 monooxygenase system
   3. The phagocytic respiratory burst
   4. Nitric Oxide (NO)
   5. Toxins, radiation, etc.
8. The hydroxyl radical is the most reactive species of ROS
9. ROS have been implicated in many diseases; the presence of ROS in different disease states does NOT necessarily prove causation of those diseases by ROS
10. Antioxidants function by (further) reducing ROS molecules, but does NOT mean they are the only possible treatments since NOT all radicals are oxygen species

Question 3

Glutathione

Answer 3

1. Struecture and function of glutathione (GLUTAmate + cysteine [THIO] + glyciNE)
2. Reduced glutathione converts hydrogen peroxide to water
3. Glutathione peroxidase regenerates oxidatized glutathione, while glutathione reductase regenerates reduced glutathione
4. Peroxide gets converted to water and oxygen
5. Take NOTE that it is cysTEine NOT methionine, although both contain thiol (SH) group
6. 2 additional H’s are required (from NADPH) in the replenishing of the glutathione

Question 4

Antioxidants

Answer 4

1. Many antioxidants are reducing agents
2. Physiologically significant antioxidants include:
   
   1. Glutathione (peroxidase)
   2. Ubiquinol (Coenzyme Q)
   3. Lipoic acid (PDH complex)
   4. Uric acid
   5. Ascorbic acid (Vitamin C)
   6. Carotenes (Vitamin A)
   7. Tocopherol (Vitamin E)
3. Melatonin is a pineal hormone, which can function as a scavenger of hydroxyl radicals; since sunlight is oxidizing, melatonin is released to recuperate
4. Supplementation of antioxidants has NOT been proven to decrease the risk of disease; supplementation wich certain antioxidants has possibly even increased the risk of disease
5. It is essential to eat a balanced diet, which would contain the necessary antioxidants

• Vitamin overdose; natural vitamin different from synthetic ones found in pills; all antioxidants are NOT the same from each other (target different things); not the most comon thing for people to be under oxidative stress; ginkgo is also anti-oxidant
• Free radical isn’t necessarily inherently bad, since it plays a role in killing germs, eliminating cancer, etc.

Question 5

Damage cuased by ROS

Answer 5

1. Mutation of DNA
2. Peroxidation of lipid (i.e. oxidation of lipid)
3. Fragmentation of proteins and cross-linking

Question 6

The Cytochrome P450 Monooxygenase

Answer 6

1. The cytochrome P450 monooxygenase system consist of 2 portions:
   
   1. Inner mitochondrial cytochrome (containing heme) P450 (CYP) is involved with the hydroxylation of steroids (cholesterol, steroid horones, vitamin D, etc.)
   2. ER associated cytochrome P450 (utilizing NADPH) is involved with the hydroxylation of xenobiotics (foreign substance that is not considered food; any medication, toxin, preservatives, anything that is foreign) making them more water soluble
2. Cytochrome P450 represents a superfamily of heme-containing proteins
3. Xenobiotic-metabolizing cytochrome P450 proteins are bound to the membranes of the smooth ER in the liver, the lungs, the small intestine, and other organs
4. If two different toxins are attacking same molecular substance and one is more efficient, that difference may be due to one staying put and one staying on very briefly in the system; CYP is involved in making xenobiotics more water soluble so better chance of getting washed away so that to minimize the period of exposure to the harmful substance inside the body.
5. If xenobiotics stick around too long, such as fat-soluble vitamin overdose, they may possible get more dangerous vs. water-soluble ones
6. Suppression/absence of certain CYP’s involved in many diseases

Question 7

Hydroxylation of xenobiotics

Answer 7

1. R (steroids or xenobiotic)
2. R-H + O₂ + NADPH+H⁺ → R-OH + H₂O + NADP+
3. This process takes place in ER
4. Hydroxylation of xenobiotics is known as Phase 1 reactions. These reactions can:
   
   1. Increase the water solubility of the chemical, facilitating its excretion in the urine and/or bile
   2. Activate a drug (pro-drug) into an active drug
      
      1. Genetic testing are done on patients to know what CYP they are missing and if they are capable of metabolizing certain drugs
   3. De-activate a drug
5. In Phase 2 reactions, the water solubility of the xenobiotic can be enhanced by conjugation with water soluble compounds as glucuronic acid, sulfate, glycine, glutamine, glutathione, and etc. (very much required to make xenobiotics really water-soluble)
6. Phase 2 reactions are essential because products of conjugation reactions have increased molecular weight and tend to be less active than their substrates, unlike Phase I reactions which often produce active metabolites.
7. NOTE: pro-carcinogens can also be activated into carcinogens (important ‘side-effect’)
8. Grape fruit juice can inhibit CYP in a way leading to drug overdose
9. Note that CYP system is NOT part of an immune response; far from it

Question 8

Related journals

Answer 8

1. Reduced effectiveness of Plavix in patients who are poor metabolizers of the drug
2. H2O2-mediated oxidative stress affects human hair color by bluding methionine sulfoxide repair
3. Extension of murine life span by overexpression of catalase targeted to mitochondria (didn’t turn out the way expected)

Question 9

Consequences of taking multiple drugs

Answer 9

1. The conjugates formed by the detoxification apparatus are sometimes more toxic than the original parent compound
2. Many of the cytochrome P450 enzymes are given to induction and also to inhibition
3. A drug may activate or deactivate CYP but the second drug may also activate or deactivate CYP; really need to know the drugs’ impact.
4. Also need to know the drug use alcohol use of the patients since they are also factors affecting the function of CYP
5. Genetic polymorphism and inhibition (activation) of CYP really make appropriate drug administration difficult
6. 7.

Question 10

Neutrophils and Monocytes

Answer 10

1. Neutrophils and monocytes employ both:
   
   1. Oxygen-independent
   2. Oxygen-dependent
   3. Processes for killing bacteria that have been introduced into them by phagocytosis
2. Oxygen independent processes involve:
   
   1. The inhibition of bacterial growth in a low pH environment
   2. The activation of anti-bacterial enzymes such as glycosylases, lysozyme, phospholipases, nucleases, etc. where none requires oxygen
3. Oxygen dependent system consists of the following steps:
   
   1. Attachment of the bacteria to the phagocytic cell through the agency of immunoglobulins
   2. Internalization of bacteria into phagosome
   3. Fusion of phagosome to a lysosome, forming a phagolysosome
   4. Respiratory burst

Question 11

Respiratory Burst

Answer 11

1. The respiratory burst utilizes superoxide, which is the product of: NADPH + 2O₂ –(NADPH oxidase)→ 2O₂^- + NADP⁺ + H⁺
2. Superoxide (spontaneously) get converted by superoxide dismutase to become peroxide and further reactions generate HOCl , hypochlorous acid (the oxidizer in bleach) by Myeloperoxidase and hydroxyl radical (OH^-)
3. The macrophage contains a high concentration of ascorbate in order to avoid oxidative damage.
4. Respiratory burst reaction steps:
   
   1. Attachment of the pathogen to a phagocytic cell
   2. Ingestion of the microorganism into a phagosome, which fuse with lysosome to become phagolysosome.
   3. Destruction of the microorganism by either of the 3 total different ways of killing bacterium: superoxide, HOCl, or hydroxyl radical
5. Individuals who have chronic granulomatous disease (difficulty forming the reactive oxygen compounds esp. superoxide) are at an increased susceptibility to infections by certain microorganisms such as Staphylococcus. This is because Staph has catalse in them, braking down hydrogen peroxide, making it virtually impossible to be destroyed, as it would have been the only source of making other ROS aside from superoxide.

Question 12

Nitric Oxide

Answer 12

1. NO is produced by the catalytic oxidation of L-Arg to L-citrullin using oxygen and NADPH as substrates carried out by NO synthase
2. There are THREE NO synthases:
   
   1. eNOS: found in endothelial cells — constitutive (synthesized at a constant rate, and is not induced or inhibited); calcium dependent
   2. nNOS: found in neurons — constitutive; calcium dependent
   3. iNOS: inducible, can function independently of calcium— found in many cell types
3. NO has a very short halflife and acts in an autocrine or paracrine manner.
4. Actions of NO include the following:
   
   1. Relaxation of smooth muscle
      
      1. Relaxation of walls of coronary arteries/arterioles
      2. Increase kidney rate of filtration
      3. Production of penile erection
      4. Inhibition of uterine smooth muscle contraction
   2. Stimulation of endocrine secretions
   3. Mediation of immunity
      
      1. Active inside macrophage lysosomes
   4. And other functions

Question 13

Glucose 6-phosphate dehydrogenase deficiency (G6PDD)

Answer 13

1. Glucose 6-phosphate dehydrogenase deficiency (G6PDD) is the most revalent human genetic enzyme deficiency; inability to generate G6PD
2. G6PDD affects more than 400 million people
3. The deficiency is X-linked (men more susceptible)
4. The deficiency is found in about 10% of all African American males
5. The deficiency impairs the ability of the cell to form the NADPH that is essential for the maintenance of the reduced (active) glutathione pool.
6. Free radicals and peroxides formed within the cells cannot be neutralized, causing denaturation of cytosolic protein (for example, hemoglobin,
   forming Heinz bodies) and membrane proteins.
7. The cells become rigid, and they are removed by the reticuloendothelial system of the spleen and liver.
8. Hemolytic anemia can be caused by the production of free radicals and peroxides following the taking of oxidant drugs, ingestion of fava beans, or severe infections.
9. Vulnerability of erythrocytes to G6PDD:
   
   1. No protein synthesis
   2. No alternative sources of NADPH; PPP is the only means of generating NADPH
   3. High O2 tension in arterial blood
   4. High levels of iron-containing heme
10. There is the risk of neonatal jaundice. This arises from the increased production of unconjugated bilirubin
11. Most individuals with G6PDD do NOT exhibit symptoms
12. However, upon exposure to certain oxidants or drugs or if infected with pathogens, they may develop hemolytic anemia (due to unstable RBC)
13. Female heterozygotes appear to have an increased immunity against malaria.
14. There is an age-related decline in erythrocyte enzyme activity, including G6PD
15. Alternative anti-oxidative systems in erythrocytes include:
    
    1. Catalase
    2. Superoxide dismutase
    3. Methemoglobin reductase
16. Heinz bodies are found in other hemoglobinapathies also
17. Heinz body-containing erythrocytes are often characterized by ROS-generated membrane deformities and are targeted for destruction by spleen macrophages
18. G6PD activity is dependent upon the age of the erythrocyte

Question 14

Clinical correlations

Answer 14

1. The determination of elevated levels of myeloperoxidase (MPO) might possibly help in the prediction of coronary artery disease in seemingly healthy individuals
2. The possible link between MPO and coronary heart disease may show the relevance of free radicals in causing heart diseases, links between some cholesterol and free radicals
3. Excess iron can also lead to free radical production; along with smoking
4. The prevalence of hypertension and diabetes mellitus appears to be relatively high in certain ethnic/racial groups.
   
   1. African Americans tend to have more diabetes mellitus (sugar-high) than other ethnic groups
   2. Hypertension and diabetes are connected ; blood pressure and PPP; NO relaxes blood vessels reducing the blood pressure; less NO, blood pressure goes up
   3. PPP lead to production of nucleotides; glycolysis and problems with PPP have to do with diabetes