Origins 1 MCQ 2- Lipid Metabolism Flashcards
1
Q
What are the essential fatty acids?
A
Linoleic and Linolenic fatty acids
2
Q
Bile acids and lipid digestion
A
Before being digested, lipids must be emulsified into micelles. This is accomplished by the addition of bile acids.
3
Q
The polyunsaturated fatty acid linolenic acid (18:3:9,12,15) is elongated by the addition of two carbons to the carbonyl end of the molecule. Where are the carbon-carbon double bonds now located?
A
Carbon double bonds at positions 11, 14, and 17.
4
Q
Where does de novo fatty acid synthesis occur compared to fatty acid degradation?
A
Synthesis: cytoplasm
Degradation: mitochondria
5
Q
Carbon dioxide and fatty acid synthesis/degradation
A
CO2 is required for fatty acid synthesis, but is not retained in the final product.
CO2 is not required for fatty acid oxidation.
6
Q
NAD+/NADPH and fatty acid synthesis/degradation
A
NADPH is the cofactors for fatty acid synthesis.
NAD+ is utilized for degradation
7
Q
Citrate and fatty acid synthesis/degradation
A
Citrate is required for fatty acid synthesis but is not utilized in oxidation
8
Q
Where does de nova synthesis of 16 carbon saturated fatty acids occur?
A
Cytoplasm
9
Q
Elongation of existing fatty acids takes place in the…
A
Mitochondria or ER
10
Q
Desaturation (carbon-carbon double bond formation) of fatty acids occurs in the
A
ER
11
Q
What is the rate limiting enzyme in fatty acid synthesis?
A
Acetyl CoA Carboxylase
12
Q
What is the reaction that produces malonyl CoA?
A
Acetyl CoA + ATP + HCO3 ——-(acetyl CoA carboxylase + biotin)—-> malonyl CoA
13
Q
Regulation of acetyl CoA carboxylase
A
Allosteric activator: citrate
Allosteric inhibitor: palmitoyl CoA
Phosphorylation:
-inactivated ACC vía cAMP protein kinase, mediated by the action of glucagon.
- inactivated ACC vía 5’ AMP mediated kinase activity (signals low energy state in cell)
14
Q
De nova fatty acid synthesis steps
A
1) 4 carbon intermediate in the FAS complex reacts with a molecule of malonyl CoA to form a 6 carbon intermediate
2) cycle keeps going of malonyl joining until palmitic acid (C16:0) is formed
3) palmitate is released from the FAS by thioesterase
- can be used to form longer chain fatty acids, other lipids, or is stored
15
Q
Overall reaction for palmitate formation
A
8 acetyl CoA + 14 NADPH + 7 ATP + 7 CO2 ——> palmitate + 8 HS-CoA + 14 NADP + 7ADP + 7 CO2 + 7 H2O + 7 P(i)
16
Q
Sources of NADPH for fatty acid synthesis
A
Liver: HMP shunt
Brain: cytoplasmic isocitrate dehydrogenase
Muscle: Malic enzyme (converts malate to pyruvate)
17
Q
Major site of fatty acid degradation is in the…
A
Mitochondria
18
Q
Cytoplasmic fatty acids
A
Must be activated by an acyl CoA synthetase (thiokinase) in order to pass into the mitochondria (where degradation occurs).
Activation resulting in formation of fatty acid CoA thioesters results in the hydrolysis of ATP to AMP.
19
Q
Transportation process of fatty acids into the mitochondrial matrix for degradation
A
1) activated fatty acids (acyl CoA) are esterified to carnitine by CPT 1.
2) carnitine acyl-translocase transfers fatty acids into mitochondrial matrix
3) once in matrix, CPT II transfers acyl fatty acids from carnitine to CoA
20
Q
Inhibition of fatty acid transport for degradation
A
Malonyl CoA inhibits CPT 1, thereby preventing the uptake of fatty acids into the mitochondria matrix during fatty acid synthesis.
21
Q
Products of beta oxidation of fatty acids
A
Acetyl CoA and acyl CoA fatty acid ester (that’s two carbons shorter).
22
Q
Cycle of beta oxidation is repeated until
A
The entire acyl CoA derivative is converted into acetyl CoA (2 carbons at a time).
23
Q
What ketone bodies are produced in the liver during beta oxidation and gluconeogenesis?
A
Acetoacetate
B-hydroxybutyrate
Acetone
24
Q
Ketone bodies:
A
1) organic acids that are produced in the liver during fasting
2) they are degraded in tissues other than liver and provide a source of ATP energy
3) ketone bodies and glucose (produced by gluconeogenesis) are both water soluble metabolites which can be used for ATP production in tissues
4) produced in great quantity when the krebs cycle is flooded with acetyl-CoA, that is, when b-oxidation is proceeding rapidly but carbohydrate oxidation is not
25
Brain’s use of ketone bodies
During prolonged fasting/starvation, the brain induces and uses beta hydroxybutyrate dehydrogenase to help compensate for the decreased availability of amino acids for gluconeogenesis
26
Degradation of polyunsaturated fatty acids (ex: linoleic acid)
Same number of acetyl CoAs formed as for saturated fatty acids of the same length, but 1.5 and 2.5 fewer molecules of ATP are made for odd and even numbered carbon-carbon double bonds, respectively.
27
Alpha Oxidation
Requires NAD+, ascorbic acid, and oxygen. It is used to help degrade branched chain fatty acids derived from chlorophyll.
28
Omega oxidation
Requires NADPH, molecular oxygen and an oxidase (mono-oxygenate) that contains cytochrome P450.
Plays a role in bile acid formation
29
Peroxisomal degradation
Used to degrade very long chain fatty acids to medium chain fatty acids. The medium then goes through beta oxidation.
30
Biotin’s role in fatty acid oxidation
Required in the oxidation of odd-chain fatty acids
31
Carnitine’s role in fatty acid oxidation
Transports fatty acid CoA thioesters into the mitochondria for oxidation
32
B12’s role in fatty acid oxidation
Requires by odd-carbon fatty acids to be completely degraded
33
FAD’s role in fatty acid oxidation
Is reduced in the first step of beta-oxidation. It’s use results in less ATP/molecule than NADH.
34
Oxidative degradation of a fatty acid begins at which end of the molecule?
Carboxyl end
35
How is ketosis beneficial?
It allows continued oxidation of fatty acids in the absence of TCA cycle function
36
Odd-chain fatty acids
Yields propionyl CoA. It’s metabolized to succinyl CoA
37
Fatty Acid elongation
This occurs in mitochondria and endoplasmic reticulum and involves the successive addition of two carbons to the carboxyl end of fatty acids to produce 18-, 20-, 22-, and 24- carbon fatty acids.
38
Essential fatty acid linoleic 18:2 is a precursor for which polyunsaturated fatty acid?
20:4 (arachidonic); Although 18:2 is normally essential, when 20:4 is obtained from the diet the shorter chain polyunsaturated fatty acid is not required.
39
Essential fatty acid linolenic 18:3 is a precursor for which polyunsaturated fatty acid?
22:6 (docosahexaenoic acids) ; Although 18:3 is normally essential, when 22:6 is obtained from the diet the shorter chain polyunsaturated fatty acid is not required.
40
Why is CO2 required for fatty acid synthesis but not incorporated into the fatty acid molecule?
The CO2 is incorporated first into malonyl CoA (where it, in effect, activates the
second carbon of acetyl CoA) and then is released again as CO2 (as the acetyl CoA moiety of malonyl CoA is joined at the carboxyl end of the growing fatty acid).
41
What coenzyme is involved in Acetyl CoA carboxylase reactions?
Biotin
42
What is the significance of acetyl CoA carboxylase stimulation by citrate in controlling fatty acid synthesis?
Increasing levels of citrate mean that the Krebs cycle is loaded. It is advantageous to convert acetyl CoA to fatty acid rather than to oxidize it immediately for energy.
43
How does intra-mitochondrial acetyl CoA serve as the starting material for cytoplasmic fatty acid synthesis?
This occurs via citrate and citrate synthase inside and citrate lyase outside mitochondria.
44
Citrate lyase enzyme converts cytoplasmic citrate to acetyl CoA and oxaloacetate. What is the fate of the cytoplasmic acetyl CoA which is formed?
Cytoplasmic acetate is used for fatty acid and steroid biosynthesis and, depending on the cell, for various isoprenoid derivatives such as cholesterol, etc.
45
Why does fatty acid synthase stop adding 2 carbon units mainly at C16:0? What happens to this saturated fatty acid formed in the synthesis?
A deacylase (thioesterase) with a specificity for C16 releases free palmitate.
46
What is the oxidizing agent in the reaction that produces the double bond in unsaturated fatty acids?
Molecular oxygen (O2) is the oxidant; however, a reducing agent (NADPH) is also required by the "mixed-function oxidase."
47
Why is an individual on an essential fatty acid deficient diet incapable of producing polyunsaturated fatty acids such as 20:4 and 22:6?
If essential fatty acids are eliminated, the body attempts to compensate by using 18:1 9 (ω9), which is not essential.The major result is the accumulation of 20:3 ω9 and 22:3 ω9 fatty acids. This is because the desaturase is not capable of adding a double bond beyond the C 9 position of a fatty acid (i.e., addition is only at three-carbon intervals toward the carboxyl end).
48
In the synthesis of a molecule of palmitate from acetyl CoA, how many ATP(s) must be expended.
Seven ATP are expended -- one for each acetyl CoA converted to malonyl CoA.
49
TRUE of FALSE?
In the course of fatty acid synthesis, the growing acyl group is transferred between an acyl-carrier protein and the condensing enzyme.
True
50
What type of reactions are inhibited by avidin?
The reaction must involve a biotin-requiring enzyme. Avidin is a protein from egg white that specifically inhibits biotin-requiring enzymes. These enzymes always catalyze ATP-
coupled fixation of CO2 as a carboxyl group.
51
Which step in β-oxidation results in the production of FADH2?
2
52
Which step occurs only once during the oxidation of fatty acids to CO2 and H2O?
1
53
If the reactions were reversed as in elongation of fatty acids which steps would utilize NADPH?
steps 4 and 2
54
During the formation of palmitic acid by de novo fatty acid synthesis how many molecules of CO2 are involved (required)?
7
55
Acetyl COA carboxylase activity is inhibited by the action of which of the following?
glucagon and palmitic acid
56
High quantities of linolenic acid in the diet lead to adipose tissue triglycerides which contain a large amount of which fatty acid?
18:3
57
Lack of insulin as in uncontrolled diabetes, would have what effect on adipose tissue triglycerides?
increased degradation
58
The amino acid serine is involved in the synthesis of which two lipids?
sphingomyelin and phosphatidylserine
59
The cellular degradation of PIP2 by phospholipase C leads to which two of the following second messengers?
DAG and IP3
60
Membrane proteins serve to function in all of the following except?
the site of glycolysis
61
Which lipoprotein particle is termed pre β-lipoprotein upon electrophoretic separation?
VLDL
62
Which lipoprotein is the first formed after ingestion of a meal containing lipid?
chylomicrons
63
Which lipoprotein leads to a decrease in HMG CoA reductase activity and the down regulation of receptors when taken up by human fibroblasts (skin cells)?
LDL
64
LCAT (lecithin cholesterol acyl transferase) aids in the formation of LDL along with lipid exchange from which of the lipoprotein particles?
HDL
65
Which enzyme’s activity leads to the formation of TXA2 in platelets?
cyclooxygenase
66
Which enzyme’s activity leads to the formation of ketone bodies in liver during fasting?
HMG CoA lyase
67
Which enzyme’s activity leads to the formation of leukotriene’s in leucocytes?
lipoxygenase
68
Which enzyme is located in the plasma membrane of cells in the capillary beds surrounding adipose tissue and muscle that degrades triglycerides in lipoproteins?
lipoprotein lipase
69
Removal of a cholesterol metabolite from the body can be used clinically to treat hypercholesterolemia. In this case therapy is directed toward the elimination of:
bile acids
70
Both the conversion of monocarboxylic acids to di-carboxylic acids and the biosynthesis
of cholic acid involve:
ω-oxidation
71
Which one of the following fatty acids becomes a “non-essential” human nutrient if arachidonic acid is included in the diet?
Linoleate
72
A genetic deficiency of which of the following enzymes would result in reduced levels of cholesterol esters in LDL lipoproteins?
Lecithin: cholesterol acyltransferase (LCAT)
73
In mammals, phosphatidylserine is formed
Exchange of ethanolamine of phosphatidylethanolamine with serine.
74
What metabolic activity would be consistent with a low circulation [insulin]:[glucagon] ratio, mild hypoglycemia, and mild keto acidosis?
increased triglyceride breakdown in adipose and decreased fatty acid synthesis in liver
75
Which reaction in the liver should be simulated most in a starving individual?
conversion of HMG CoA to acetoacetic acid
76
Biotin binds tightly to a protein in egg white called avidin. What reaction would be most likely to be inhibited if excess avidin were present?
propionyl CoA carboxylase
77
When one takes aspirin, prostaglandin synthesis is lost in platelets during their lifetime because:
the cell does not have a nucleus to resynthesize new enzyme.
78
Blockage of the common bile duct will result in all of the following EXCEPT:
A. elevated serum amylase and lipase activity
B. a lack of bile acids for micelle formation in the intestine
C. elevated chylomicron levels in blood
D. premature activation of phospholipase A2 in the pancreas
E. inhibition of bile acid formation in liver
C. elevated chylomicron levels in blood
79
The distinctive feature of a ganglioside is that it is a glycosphingolipid containing
N-acetylneuraminic acid (sialic acid)
80
Radioactive carbon dioxide is used to form malonyl CoA from acetyl CoA. If the labeled malonyl CoA is used for palmitate synthesis which position(s) in the fatty acid would be labeled?
None of the carbons.
81
During the digestion, absorption, transport and storage of dietary triacylglycerol, the ingested triacylglycerol is subjected to the following processes:
1. ) emulsification with the aid of bile acids
2. ) hydrolysis by pancreatic lipase
3. ) resynthesis from 2-monoacylglycerol and acyl CoA
4. ) formation of chylomicrons
5. ) action of lipoprotein lipase
82
A protein kinase can phosphorylate and activate which of the following?
hormone sensitive lipase
83
Which of the following substances is found as a part of phosphatidylcholine and sphingomyelin?
phosphocholine
84
Chylomicrons are:
produced in the intestine, and contains triglycerides, cholesterol, cholesterol
esters, protein, phospholipids.
85
n the absence of glucose uptake in adipose tissue there is:
Decreased triglycerides synthesis because of a lack of glycerol phosphate.
86
All of the following are true about LDL-particles EXCEPT which one?
A. They carry a high concentration of cholesterol esters.
B. Apo-A is their major apolipoprotein.
C, They enter cells via a specific membrane receptor.
D. They are degraded by intracellular lysosomal enzymes.
E. The plasma of patients with familial hypercholesterolemia contains high amounts.
B. Apo-A is their major apolipoprotein.
87
Carnitine acyltransferase I catalyzes the:
transfer of a fatty acyl group from fatty acyl-CoA to carnitine.
88
Which of the following is most likely to carry free fatty acids and bile acids in blood?
albumin
89
Formation of diacylglycerol from phosphatidylcholine is due to the action of:
phospholipase C.
90
Which of the following substances prevents platelet aggregation and is a potent vasodilator?
prostacyclin
91
The direct utilization of SAM for lecithin synthesis requires:
a source of phosphatidylethanolamine
92
In prostaglandin pathways
inhibition of platelet cyclooxygenase results in deficiency in TXA2.
93
Which of the following is an immediate product of phosphatidic acid phosphatase (phospholipase C) catalysis and, by a different route, an activator of protein kinase C?
1, 2-diacylglycerol
94
The first committed step in fatty acid biosynthesis is catalyzed by
acetyl CoA carboxylase
95
The sequence of reactions for the beta-oxidation of fatty acids is
activation, dehydrogenation, hydration, dehydrogenation and cleavage.
96
```
Glucose is used by mammals for all of the following purposes EXCEPT
A. linoleic acid synthesis.
B. oxaloacetate synthesis.
C. glycerol phosphate synthesis.
D. ribose 5-phosphate synthesis.
E. reactions that generate NADPH.
```
linoleic acid synthesis.
97
In humans, reverse cholesterol transport (transport of tissue cholesterol back to the liver) is expedited by which lipoprotein particle?
HDL
98
Your patient stops for an “Egg McMuffin” on his way to a “fasting” serum lipoprotein electrophoresis. Which lipoprotein will be elevated ~3 hours later?
chylomicron
99
An increased synthesis of acetoacetate and beta-hydroxybutyrate by the liver
is sustained by increased triacylglycerol hydrolysis in adipose tissue.
100
How many of the carbon atoms from stearic acid may contribute, through cellular metabolism, to the net synthesis of glucose?
0
101
The oxidation to carbon dioxide and water of a cytoplasmic 14 carbon saturated fatty acid (myristic acid) should result in a NET gain of how many high energy phosphates?
92
102
Oxidation of acetoacetic acid to carbon dioxide and water
requires transfer to the CoA residue of succinyl CoA.
103
With respect to lipoprotein metabolism
lipoprotein lipase is activated by apolipoprotein C.
104
A diabetic patient develops Type IV hyperlipidemia, characterized by elevated concentrations of VLDL. The most likely explanation for this is:
fatty acids delivered to the liver in excess of those used for beta-oxidation
are incorporated into triglycerides of VLDL.
105
Hormone sensitive lipase and lipoprotein lipase are similar in that they both:
serve as a means of supplying free fatty acids for oxidative metabolism.
106
All of the following statements about Type I hyperlipoproteinemia are correct EXCEPT?
Manifestations of the disease are first observed in adults.
107
Which of the following is produced in the liver and only in the liver?
glycocholic acid
108
The conversion of propionyl CoA to succinyl CoA
occurs with both odd-numbered-and branched chain fatty acids.
109
Which of the following accumulates in tissues of patients with Niemann-Pick disease?
Sphingomyelin
110
Plasma electrophoresis from a patient suspected of having hyperlipoproteinemia has an elevated density of pre b-lipoproteins. This is the same as which lipoprotein?
VLDL
111
In Type 2 A hyperlipoproteinemia the cellular receptor for which lipoprotein is defective, or is expressed at low levels, in human fibroblasts (skin cells)?
LDL
112
Plasma LDL isolated from a patient with a suspected enzyme deficiency exhibits reduced levels of cholesterol esters and elevated free (unesterified) cholesterol. To confirm the diagnosis one should test the activity of which enzyme?
Lecithin: cholesterol acyltransferase (LCAT)
113
In patients the course of pancreatitis arising from blockage of the common bile duct can
best be followed by determining:
serum amylase and lipase activity
114
Following a meal containing fats, short chain fatty acids and bile acids would be transported via the portal circulation to the liver by?
albumin
115
Prostaglandins
- consist of 20 carbon polyunsaturated fatty acid derivatives (eicosanoids), formed from arachidonic acid
- plays an important role in the blood clotting process
116
What is the rate limiting step in prostaglandin synthesis?
cyclooxygenase
117
What are the substrates for cyclooxygenase?
Polyunsaturated fatty acids released from membrane lipids by phospholipases; cyclooxygenase can utilize ω6 (20:3, 20:4) or ω3 (20:5) fatty acids in prostaglandin formation
118
What is an inhibitor of prostaglandin synthesis?
Aspirin. Aspirin is very effective at inhibiting COX-I, a constitutive form of the enzyme, but it also inhibits COX-II, an inducible form often expressed during inflammation.Only aspirin is recommended to prevent platelet clumping.
119
Prostaglandin degradation
is relatively rapid and involves reduction of the trans-13, 14 carbon double bond and formation of a 15-keto derivative. Prostaglandins are then somewhat altered by both omega- and beta- fatty acid oxidation and ultimately the derivatives appear in the urine.
120
thromboxanes
- A sub-class of eicosanoids that are extremely effective in causing blood platelets to clump
-Thromboxane
synthesis involves COX I
121
TXA2
-About 95% of all prostaglandins formed in platelets
is TXA2.
- Vascular constriction may also be enhanced by TXA2.
122
PGI2
- produced by the Arterial endothelial cells
- raises platelet cAMP levels and prevents platelet clumping
- appears to relax vascular smooth muscle
123
Leukotrienes
- class of eicosanoid derivatives is involved in inflammation and enhancing vasoconstriction
- cause a slow but prolonged contraction of smooth muscle in the airways and gastrointestinal tract
124
Singulair
a receptor antagonist that blocks the action of leukotrienes containing cysteine e.g.
LTC4, by acting on target tissues
125
Zileuton
- directly inhibits the enzyme 5-lipoxygenase; - inhibition of 5-lipoxygenase may have benefits in addition to its action in treating asthma.
126
Preferred lipoxygenase substrate
is arachidonic acid, but Lipoxygenases can also use docosahexaenoic acid (DHA)
127
What is the effect of endoperoxide metabolism (e.g. PGH2) on thromboxanes in platelets?
Endoperoxides cause platelet aggregation, by conversion to thromboxanes, which is prevented by aspirin and related compounds.
128
How do the thromboxanes differ from the prostaglandins in terms of structure, site of synthesis, and mechanism of action?
They have a six-member oxygen-containing ring which is, in some cases, oxygen bridged. They are synthesized in platelets, thought to be more active and short-lived than the prostaglandins, and are effective in causing platelet clumping.
129
What is the mechanism of action of aspirin?
Aspirin appears to act by the inhibition of prostaglandin synthesis prior to the formation of the endoperoxide, by acetylating a serine residue in cyclooxygenase.
130
What is the nature of the binding of lipid to protein in the plasma lipoproteins? What types of reagents can break this binding?
Binding is primarily hydrophobic. Lipids can be extracted with organic solvents or detergents.
131
What are the main products of the action of pancreatic lipase on triglycerides?
Free fatty acids and β-monoglycerides.
132
In what form and by what route are dietary fatty acids delivered to the blood stream? What tissues then take them up? What is the role of lipoprotein lipase?
Dietary fatty acids are delivered to the blood stream primarily as triglycerides in chylomicrons in the intestinal lymph via the thoracic duct. They are taken up by the liver, muscle, and adipose tissue. Lipoprotein lipase hydrolyzes these triglycerides to free fatty acids, a step necessary for uptake by the adipose tissue and muscle.
133
What features of lipid metabolism are distinctive to:
a. adipose tissue
b. the liver
a. Hormone sensitive lipase, absence of glycerol kinase, a resulting close dependence of triglyceride turnover on carbohydrate availability and on insulin and other hormones.
b. Synthesis of plasma lipoproteins and of ketone bodies, synthesis and conjugation of bile acids, synthesis of cholesterol under feedback regulation by dietary cholesterol.
134
What conditions promote the mobilization of fat from adipose tissue? What is the principal regulation? In what form is the mobilized fat transported?
Fat mobilization is promoted by a low level of available carbohydrate; by several hormones, especially glucagon, epinephrine, and norepinephrine; and strongly inhibited by insulin. The main regulators are the hormone-dependent lipase and the availability of glycerol-3-phosphate. Mobilized fat is transported mainly as albumin-free fatty acid complexes.
135
What function does LDL serve?
LDL is the chief carrier of cholesterol in serum. It also depresses cholesterol synthesis in cells.
136
What physiological condition would be expected to result in high levels of chylomicrons?
Shortly after the ingestion of a fatty meal. Conversely, low levels of chylomicrons are found when the organism is in the fasting state.
137
What is the site of lipoprotein synthesis?
The liver synthesizes VLDL and HDL while chylomicrons are formed in the intestine. LDLs are formed in the blood from liver derived VLDL particles.
138
Leptin
- involved in the appetite control system (targets the acurate nucleus in the hypothalamus of brain)
- released when fat storage (in adipose tissue) is sufficient, and signals the brain to “stop eating”. When leptin levels increase, NPY levels fall (increases appetite).
- activates AMPK which promotes energy producing pathways (fat mobilization and oxidation) and down-regulates fatty acid synthesis (energy storage).
- makes liver and muscle cells more sensitive to insulin
139
Adiponectin
-sensitizes organs to the effects of insulin
140
Resistin
block the action of insulin in adipocytes
141
Atherosclerosis
condition in which lipid rich plaques build up in arteries throughout the body
142
Primary Risk Factors for arterial disease
↑ LDL Cholesterol Hypertension
| Smoking
143
Secondary Risk Factors for arterial disease
- ↑ TG
- diabetes
- obesity
- ↓ physical activity
- stress
- over conscientious personality
144
C-reactive protein (CRP)
- marker of systemic inflammation and a potential indicator of heart disease.
- It is produced in the liver in response to interleukin 6, a cytokine formed during vascular injury.
145
CRP levels
increase during inflammation.
146
CRP can bind to LDL particles
increase their ingestion by macrophages, leading to increased foam cell formation.
147
Decreased levels of CRP
- Aspirin and statins both lead to decreases in CRP levels
| - Having low LDL and CRP levels can lower the risk of heart attack by as much as 65%
