Figure-1- showing ATP Synthase complex.
ATP synthase complex– is embedded in the inner membrane, together with the respiratory chain complexes .The enzyme complex consists of an F0 sub complex which is a disk of “C” protein subunits. Attached is a Υ subunit in the form of a “bent axle.” Protons passing through the disk of “C” units cause it and the attached Υ subunit to rotate. The Υ subunit fits inside the F1 sub complex of three α and three β subunits, which are fixed to the membrane and do not rotate. The flow of protons through F0 causes it to rotate, driving the production of ATP in the F1 complex.
ii) Visual Cycle
In the retina, retinaldehyde functions as the prosthetic group of the light-sensitive opsin proteins, forming rhodopsin (in rods) and iodopsin (in cones). Any one cone cell contains only one type of opsin, and is sensitive to only one color.
In the pigment epithelium of the retina, all-trans-retinol is isomerized to 11-cis-retinol and oxidized to 11-cis-retinaldehyde. This reacts with a lysine residue in opsin, forming the holoprotein rhodopsin .
The absorption of light by rhodopsin causes isomerization of the retinaldehyde from 11-cis to all-trans, and a conformational change in opsin. This results in the release of retinaldehyde from the protein, and the initiation of a nerve impulse.
The formation of the initial excited form of rhodopsin, bathorhodopsin, occurs within picoseconds of illumination. There is then a series of conformational changes leading to the formation of metarhodopsin II, which initiates a guanine nucleotide amplification cascade and then a nerve impulse. The final step is hydrolysis to release all-trans-retinaldehyde and opsin. The key to initiation of the visual cycle is the availability of 11-cis-retinaldehyde, and hence vitamin A. In deficiency, both the time taken to adapt to darkness and the ability to see in poor light are impaired.
Figure-2- Showing visual cycle
iii) Carnitine shuttle
Figure-3- Showing carnitine shuttle.
Carnitine – (CH3)3N+—CH2—CH(OH)—CH2—COO–, is widely distributed and is particularly abundant in muscle.
Long-chain acyl-CoA (or FFA) will not penetrate the inner membrane of mitochondria. However, carnitine palmitoyl transferase-I, present in the outer mitochondrial membrane, converts long-chain acyl-CoA to acylcarnitine, which is able to penetrate the inner membrane and gain access to the Β-oxidation system of enzymes (Figure -3).
Carnitine-acyl carnitine translocase acts as an inner membrane exchange transporter. Acyl carnitine is transported in, coupled with the transport out of one molecule of carnitine. The acyl carnitine then reacts with CoA, catalyzed by carnitine palmitoyl transferase-II, located on the inside of the inner membrane. Acyl-CoA is re-formed in the mitochondrial matrix, and carnitine is liberated.
This carnitine shuttle also serves to prevent uptake into the mitochondrion (and hence oxidation) of fatty acids synthesized in the cytosol in the fed state; malonyl CoA (the precursor for fatty acid synthesis) is a potent inhibitor of carnitine palmitoyl transferase I in the outer mitochondrial membrane.
Short and medium chain fatty acids do not require carnitine for their transportation across the inner mitochondrial membrane.
Carnitine deficiency may cause muscle necrosis, myoglobinuria, lipid-storage myopathy, hypoglycemia, fatty liver, and Hyperammonemia with muscle aches, fatigue, confusion, and cardiomyopathy.
Q.3- a) Give a brief account of the role of niacin as a coenzyme, highlight the important reactions and mention the names of the enzymes requiring niacin as a coenzyme. What would be the clinical implications of its deficiency?
Answer- The term niacin refers to both nicotinic acid and its amide derivative, nicotinamide (niacinamide) Both are used to form the coenzymes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+).
Niacin is a member of the water-soluble B- vitamin complex. The amino acid tryptophan can be converted to nicotinic acid in humans; therefore niacin is not really a vitamin provided that an adequate dietary supply of tryptophan is available. Some 60 mg of tryptophan is equivalent to 1 mg of dietary niacin.
Redox reactions – As many as 200 enzymes require the niacin coenzymes, NAD+ and NADP+, mainly to accept or donate electrons for redox reactions.
NAD+ functions most often in energy producing reactions involving the degradation (catabolism) of carbohydrates, fats, proteins, and alcohol.
NADP+ functions more often in biosynthetic (anabolic) reactions, such as in the synthesis of all macromolecules, including fatty acids and cholesterol.
Some of the important enzymes requiring NAD+ and NADP+ are as follows-
NAD+ dependent enzymes –
1) Glyceraldehyde-3-phosphae dehydrogenase
2) Pyruvate dehydrogenase complex
3) Mitochondrial Isocitrate dehydrogenase
4) Alpha keto glutarate dehydrogenase complex
5) Malate dehydrogenase
6) Lactate dehydrogenase
7) Beta hydroxy acyl co A dehydrogenase
8) Cytosolic glycerol-3-phosphate dehydrogenase
NADPH dependent enzymes
1) HMG co A reductase
2) Enoyl reductase
3) Keto acyl reductase
4) Dihydrofolate reductase
5) Met hemoglobin reductase
6) Ribonucleotide reductase
Non-redox reactions –
The niacin coenzyme, NAD+, is the substrate (reactant) for two classes of enzymes-
(mono-ADP-ribosyltransferases and poly-ADP-ribose polymerase) that separate the niacin moiety from NAD+ and transfer ADP-ribose to proteins.
1) Mono-ADP-ribosyltransferases –These enzymes and their products, ADP-ribosylated proteins, are thought to play a role in cell signaling by affecting G-protein activity. G-proteins are proteins that bind guanosine-5′-triphosphate (GTP) and act as intermediaries in a number of cell-signaling pathways.
2) Poly-ADP-ribose polymerases (PARPs) are enzymes that catalyze the transfer of many ADP-ribose units from NAD+ to acceptor proteins. PARPs appear to function in DNA repair and stress responses, cell signaling, transcription, regulation or apoptosis, chromatin structure, and cell differentiation, suggesting a possible role for NAD+ in cancer prevention
3) A third class of enzymes (ADP-ribosyl cyclase) catalyzes the formation of cyclic ADP-ribose, a molecule that works within cells to provoke the release of calcium ions from internal storage sites and probably also plays a role in cell signaling.
Niacin deficiency causes -Pellagra
Pellagra is clinically manifested by the 4 D’ s: photosensitive dermatitis, diarrhea, dementia, and death.
The early symptoms of pellagra include loss of appetite, generalized weakness, irritability, abdominal pain and vomiting.
Later symptoms are bright red glossitis, chronic or recurrent diarrhea (watery, but occasionally bloody), which leads to a state of malnutrition and cachexia.
The characteristic skin rash is characterized by pigmentation and scaling, particularly involving the sun exposed areas. Pellagra can affect any part of the body surface, but it more frequently appears in certain areas. The usual sites are the dorsal surfaces of the hands, face, neck, arms, and feet.
Figure-4- showing rashes in the sun exposed region in Pellagra As pellagra advances, neuropsychiatric symptoms such as photophobia, asthenia, depression, hallucinations, memory loss, and psychosis begin. The patient become disoriented, confused, and delirious; stupor and death result if untreated.
Causes – Pellagra is the late stage of severe niacin deficiency.
- Primary pellagra results from inadequate nicotinic acid (ie, niacin) and/or tryptophan intake in the diet.
- Secondary pellagra occurs when adequate quantities of niacin are present in the diet, but other diseases or conditions interfere with its absorption and/or processing. Such conditions include the following:
- Prolonged diarrhea
- Long-term alcoholism
- Chronic colitis
- Ileitis terminalis
- Cirrhosis of the liver
- Tuberculosis of the GI tract
- Malignant carcinoid tumor
- Hartnup syndrome
Isoniazid induced niacin deficiency: Isoniazid causes a depletion of Pyridoxal phosphate, that is required for the production of niacin from tryptophan.
Treatment -Because multiple deficiencies are common, a balanced diet, including other B vitamins (particularly riboflavin and pyridoxine), is needed. Nicotinamide is usually used to treat deficiency, because nicotinamide, unlike nicotinic acid (the most common form of niacin), does not cause flushing, itching, burning, or tingling sensations. Nicotinamide is given in doses ranging from 40 to 250 mg/day in divided doses 3 to 4 times a day.
Niacin (nicotinic acid) in large amounts is sometimes used to lower LDL cholesterol and triglyceride levels and to increase HDL cholesterol levels.
b) Give a brief description of the abnormalities that can lead to fatty liver formation. Support your answer by a suitable flow chart or a diagram summarizing the mechanisms involved in fatty liver formation.
Fatty liver (steatosis) – It is an abnormal accumulation of certain fats (triglycerides) inside liver cells. Hepatic triacylglycerol synthesis provides the immediate stimulus for the formation and secretion of VLDL. Impaired VLDL formation or secretion leads to nonmobilization of lipid components from the liver, resulting in fatty liver.
Causes of fatty liver- Imbalance in the rate of triacylglycerol formation and export causes fatty liver. For a variety of reasons, lipid—mainly as triacylglycerol—can accumulate in the liver. Extensive accumulation is regarded as a pathologic condition. When accumulation of lipid in the liver becomes chronic, fibrotic changes occur in the cells that progress to cirrhosis and impaired liver function.
Fatty livers fall into two main categories-
A) More synthesis of Triglycerides or
B) Defective VLDL synthesis (Metabolic block)
A) More synthesis of Triglycerides-Triglycerides are synthesized in excess due to more availability of Fatty acid and glycerol.
The fatty acids used are derived from two possible sources:
(1) synthesis within the liver from acetyl-CoA derived mainly from carbohydrate (perhaps not so important in humans) and (2) uptake of free fatty acids from the circulation.
The first source is predominant in the well-fed condition, when fatty acid synthesis is high and the level of circulating free fatty acids is low. As triacylglycerol does not normally accumulate in the liver under this condition, it must be inferred that it is transported from the liver in VLDL as rapidly as it is synthesized and that the synthesis of apo B-100 is not rate-limiting.
Free fatty acids from the circulation are the main source during starvation, the feeding of high-fat diets, or in diabetes mellitus, when hepatic lipogenesis is inhibited. Thus high carbohydrate diet stimulates de novo fatty acid synthesis by providing excess of Acetyl CoA and high fat feeding provides more flux of fatty acids from the diet that can be esterified to provide excess triglycerides.
B) Defective VLDL synthesis –The second type of fatty liver is usually due to a metabolic block in the production of plasma lipoproteins, thus allowing triacylglycerol to accumulate. Theoretically, the lesion may be due to-
(1) A block in apolipoproteins synthesis-
Causes- can be-
a) Protein energy Malnutrition
b) Impaired absorption
c) Presence of inhibitors of endogenous protein synthesis e.g.- Carbon tetra chloride, Puromycin, Ethionine etc.
The antibiotic puromycin, ethionine (α-amino-γ-mercaptobutyric acid), carbon tetrachloride, chloroform, phosphorus, lead, and arsenic all cause fatty liver and a marked reduction in concentration of VLDL (Figure-5). The action of ethionine is thought to be caused by a reduction in availability of ATP due to its replacing methionine in S-adenosylmethionine, trapping available adenine and preventing synthesis of ATP.
d) Hypobetalipoproteinemia- Defective apo B gene can cause impaired synthesis of apo B protein.
(2) A failure in provision of phospholipids that are found in lipoproteins-
a) A deficiency of choline, which has therefore been called a lipotropic factor can cause impaired formation of phosphatidyl choline (Lecithin),a glycerophospholipid (Figure-5)
b) Choline is formed by methylation from ethanolamine, with S-Adenosyl Methionine acting as a methyl group donor. Methionine deficiency can cause impaired choline synthesis and thus fatty liver besides other clinical defects.
c) Deficiency of essential fatty acids– can also lead to impaired Phospholipid synthesis
(3) Impaired Glycosylation- Orotic acid causes fatty liver; it is believed to interfere with glycosylation of the lipoprotein, thus inhibiting release, and may also impair the recruitment of triacylglycerol to the particles. In conditions of orotic aciduria (disorder of pyrimidine nucleotide biosynthesis), fatty liver can be observed (Figure-5)
4) Impaired secretion of VLDL- oxidative stress is a common cause for membrane disruption of lipoprotein. The action of carbon tetrachloride probably involves formation of free radicals causing lipid peroxidation (Figure-5). Some protection against this is provided by the antioxidant action of vitamin E-C, beta carotene and selenium in the supplemented diets.
Figure-5- Showing the biochemical basis of fatty liver disease. Imbalance in the rate of triacylglycerol formation and export causes fatty liver.
Clinical conditions causing fatty liver
Clinically fatty liver is of two types-
1) Non alcoholic fatty liver- Fatty liver (with or without fibrosis) due to any condition except alcoholism is called nonalcoholic steatohepatitis (Macro vesicular steatosis). Causes of nonalcoholic steatosis or NAFLD are
- Diabetes mellitus
- Drugs- corticosteroids, amiodarone, diltiazem, tamoxifen, highly active antiretroviral therapy
- Poisons (carbon tetrachloride and yellow phosphorus)
- Endocrinopathies such as Cushing’s syndrome and hypopituitarism, hypobetalipoproteinemia and other metabolic disorders,
- Obstructive sleep apnea,
2) Alcoholic fatty liver- Alcoholism leads to fat accumulation in the liver, hyperlipidemia, and ultimately cirrhosis. The fatty liver is caused by a combination of impaired fatty acid oxidation and increased lipogenesis, which is thought to be due to changes in the [NADH]/[NAD+] redox potential in the liver, and also to interference with the action of transcription factors regulating the expression of the enzymes involved in the pathways.
Oxidation of ethanol by alcohol and aldehyde dehydrogenase leads to excess production of NADH (Figure-6)
A) Effect of excess NADH – More triglyceride synthesis
1) The NADH generated competes with reducing equivalents from other substrates, including fatty acids, for the respiratory chain, inhibiting their oxidation and causing increased esterification of fatty acids to form triacylglycerol, resulting in the fatty liver.
2) Accumulation of NADH causes more formation of Glycerol-3-P (shift of equilibrium of reaction), that can be used for the synthesis of triglycerides.
B) Oxidation of ethanol, leads to the formation of acetaldehyde, which is oxidized by aldehyde dehydrogenase, producing acetate. Acetate is converted to Acetyl coA and there is more fatty acid synthesis.
C) Improper apo- protein synthesis – Malnutrition is a common finding in chronic alcoholism There is less availability of essential amino acids.
D) Impaired Phospholipid synthesis-Due to malnutrition there is less availability of essential fatty acids and choline leading to defective Phospholipid synthesis.
E) Impaired secretion of VLDL- chronic alcohol consumption is associated with oxidative stress that can cause impaired VLDL secretion.
Thus multiple factors are responsible for alcoholic fatty liver disease
Lipotropic agents- Agents such as choline, Inositol, Methionine and other essential amino acids, essential fatty acids, anti oxidant vitamins, vitamin B12, folic acid and synthetic antioxidants which have the apparent effect of removal of fats from the liver cells, and thus prevent the formation of fatty liver are called lipotropic agents.
Figure-6- Showing steps of metabolism of alcohol
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