What are ways to make a definitive diagnosis of gout?
-Identification of MSU crystals in synovial fluid leukocytes-Identification of monosodium urate crystals from a tophus
What is the purpose of synovial fluid?
lubricant and adhesive, nutrients for cartilage
What do you do with the synovial fluid?
Observe gross appearance, order cell count, order culture and gram stain, order crystal analysis (fresh unstained wet sediment)
What are the crystals in pseudogout?
What are the characteristics of normal synovial fluid?
Transparent clear or light yellow, slightly viscous, WBCs low with few or no RBCs, low percentage of neutrophils, glc similar to plasma, no crystals, neg. culture
What are the characteristics of gout fluid?
Turbid opaque fluid, WBC count between 10,000-50,000 (most neutrophils), negative culture and gram stain, MSU crystals (5-20 um needle shaped, negatively birefringent-yellow when parallel to compensator, blue when perpendicular)
What are other factors that can contribute to gout?
Hypertension, thiazide (loop diuretics), obesity, high alcohol intake, high meat intake, hyperinsulinemia, metabolic syndrome
What are some possible treatments for gout?
1. hypoxanthine to xanthine pathway inhibitors 2. xanthine to uric acid pathway inhibitors 3. diet low in purines 4. regulate genes (SLC2A9) responsible for uric acid exretion 5. slighly modify pH of blood-higher pH favors solubility
How much oxidative energy requirement for the liver, kidneys, heart, and resting skeletal muscle comes from fatty acid oxidation?
In general, what are the steps of lipid metabolism?
1. TAGs hydrolyzed in adipose tissue to fatty acids plus glycerol (via lipases) 2. Transport of fatty acids in blood to the tissues 3. Activation of fatty acids as a CoA ester 4. Transport into mitochondria via carnitine shuttle 5. Metabolism to acetyl CoA
Where do we get the fat that we burn?
Diet, liver, adipose, blood, muscle (more in trained)
How are fats from the diet transported?
How are TAGs from the liver transported?
How are free fatty acids released from adipose tissue carried in the blood?
Use plasma albumin
What hormones can trigger lipolysis? What pathway do they activate?
Adrenaline, glucagon, and ACTH---activate cAMP, which activates the lipase
Fats are the major source of energy for all human tissues except what two things?
RBCs and brain
What are the two 'oxidative machines'?
mitochondira and peroxisomes
How do fatty acids come into the mitochondria?
Acyl CoA synthetase on the outer mito membrane turns a fatty acid into an acylCoA, which can cross the outer mito membrane. The carnitine acyltransferase I (also in OMM) turns the acylCoA into acylcarnitine. The acylcarnitine crosses the IMM with the help of carnitine acylcarnitine transferase. Once in the mito matric, carnitine acyltransferase II turns the acyl carnitine back into acylCoA.
What number of carbons are considered a long chain fatty acid? Where does the catabolism take place? Very long chain?
Long chain fatty acids are 12-20 carbons. Their catabolism occurs in the mitochondria via the carnitine shuttle. The very long chain fatty acids are greater than 20 carbons and are broken down in the peroxisome.
Beta oxidation is the same for odd # fatty acid chains as it is for even until the final 3 carbons. What happens to the propionyl CoA?
Propionyl CoA is converted to methylmalonyl CoA (by adding a CO2, uses biotin and ATP). methylmalonyl CoA is converted to succinyl CoA by methylmalonyl CoA mutase (uses coenzyme form of B12).
What is methylmalonic acidemia? Methylmalonic aciduria?
Acidemia: mutase missing (can't convert methylmalonyl CoA to succinyl CoA). Aciduria: unable to convert vitamin B12 to coenzyme form. They both block the same step and result in metabolic acidosis and developmental retardation.
What type of oxidation do branched fatty acids use? Where does this occur?
alpha oxidation because beta site is inaccessible. peroxisomes
Is malonyl CoA an activator or inhibitor of the carnitine shuttle?
Inhibitor! It inhibits carnitine acyltransferase I and blocks entry of acyl groups into mito.
In case of starvation, the liver can convert excess acetyl CoA into what?
Ketone bodies. Made from HMG CoA (like cholesterol) HMG CoA lyase makes acetoacetate from HMG CoA. This acetoacetate can then make acetone or 3-hydroxybutyrate
What is Jamaican vomiting sickness?
Caused by unripe fuit which contains a toxin, hypoglycin, which inhibits medium and short chain acyl CoA dehydrogenases (inhibits B oxidation)
What is the result of carnitine deficiency?
muscle aches and weakness following exercise, elevated blood free fatty acids. nonketotic hypoglycemia because gluconeogenesis cannot be suuported by fat oxidation
What is Zellweger syndrome?
absence of peroxisomes in the liver and kidneys leads to accumulation of very long chain fatty acids in the brain
Inborn errors of metabolism are often what two types of inheritance?
autosomal recessive and X linked
PKU is a deficiency of what enzyme? What are the consequences? What other enzyme deficiency can produce the same symptoms?
PKU: phenylalanine hydroxylase deficiency, causes a build up of Phe that can't be converted to Tyrosine and also causes a buildup of toxic metabolites (2-hydroxyphenylacetic acid, phenylpyruvic acid, phenyllactic acid)--deficiency of dihydropteridine reductase (produces the cofactor for Phe phydroxylase) can give same symptoms
What are the molecular bases of enzyme deficiency?
-dysfunctional protein (hypomorphic mutation), no enzyme synthesized (null mutation), deficient co-factor processing, biotinidase deficiency, deficient activator protein, defective trans acting factor (transcription factor)
What is the function of biotinidase?
Used to convert biocytin into free biotin, which is used in many carboxylase reactions
What are the standards of diagnosis?
1. Metabolite pattern for enzyme deficiency 2. Demonstrate enzyme deficiency 3. Demonstrate mutation in gene
What are some laboratory blood tests for preliminary diagnosis of inborn metabolism errors?
What are the biochemical/molecular genetic approach to diagnosis?
Metabolite profile, demonstrate enzyme deficiency, and demonstrate mutation
What are some characteristics of diseases with decifient fatty acid oxidation?
Severe hypoglycemia/poor ketogenesis, sudden infant death, exercise intolerance, heart disease, fatty liver, severe illness (HELLP, AFLP) late in pregnancy
What is the transcription factor responsible for the regulation of fatty acid oxidation and how is it activated?
PPAR is activated by the binding of ligands, FA, and fibrates. PPAR forms a complex with RXR and this recruits coactivators. Gene transcription for fatty acid transport and oxidation increases.
The mitochondrial trifunctional protein is made of what two subunits?
HADHA-alpha subunit=enoyl CoA hydratase/LCHAD activity and HADHB-beta subunit=ketoacyl CoA thiolase activity
What is HELLP? What is it caused by?
hemolysis elevated liver enzymes, low platelets. caused by LCHAD deficiency/trifunctional protein
What are some therapies for fatty acid oxidation disorders?
Mostly nutritional, some drugs, stabilize day to day, avoid catabolic events
What is the most common fatty acid oxidation disorder?
What is the biochemical basis of clinical diagnosis of a fatty acid oxidation disorder?
acylcarnitines and urinary organic acids
What are some metabolic concepts for preventing disease episodes for a fatty acid oxidation disorder?
-avoid fasting/exercise/catabolic stress, short chain fatty acids (bypass the block), odd chain fatty acids (anaplerotic), IV glc/fluids
How are primary bile acids converted into secondary bile acids?
by bacteria in the intestine
What type of cells synthesize bile acids? How many carbons are bile acids/salts?
synthesized in liver parenchymal cells--24 carbons
What is the rate limiting enzyme of the synthesis of bile acids and what does it do?
microsomal 7-alpha-hydroxylase (CYP7A1), which installs the OH at position 7 using P450
How many grams of bile acids pass from the bile duct to the intestine each day? What percent is lost in the feces?
30 g/2% lost
How are gall stones formed?
from bile supersaturated with cholesterol
How many carbons do the steroid hormones, corticosteroids, androgens, and estrogens contain?
C21, C19, C18
What are the two types of steroid hormone receptors?
Type 1: cytoplasmic receptors Type 2: Nuclear receptors
What is the mode of action of steroid hormones binding to a cytoplasmic receptor?
Upon steroid binding to receptor, dimerization of receptor occurs and a nuclear localization signal is exposed, complex enters nucleus and binds specific response element, or hormone response element, this functions as a sequence-specific transcription factor
What is the main differences between the glucocorticoid receptor zinc finger and the standard zinc finger?
glucocorticoid has 4 Cys (vs 2 Cys and 2 His)
What is the function of zinc finger proteins?
recognize short palindrome sequences of DNA
T/F Steroid receptor protein motifs are conserved.
What type of cells release insulin? glucagon?
GABI: glucagon released by alpha cells, beta cells release insulin (both part of islets of langerhands in the pancreas)
What is a good marker for beta cell function?
C peptide! because it is equimolar to insulin conc
What is the process of insulin secretion?
Glc enters pancreatic beta cell, phosphorylated by glucokinase or hexokinase to make G6P...ultimately increases ATP which blocks the K+ channel, this depolarizes the cell and opens the Ca2+ channel. Ca2+ increase in the cell causes insulin secretion
What is the second phase of insulin release?
involves new synthesis of insulin-responds to an increase in cytosolic long acyl CoA (sign of well fed state)