chem unit 4 summer

bio catalysts

large proteins, lowers activation energy, unchanged by reaction

properties of enzymes

not permanently altered or consumed in rxn, effective in small amounts, accelerates the rate the rxn eq rate without altering eq point

enzyme rxn

E + S with ES= E+P

isoform

diff forms of the same protein

isoenzyme

enzyme isoforms that catalyze the same rxn but differ in structure and tissue distribution

zymogen= proenzyme

secreted in inactive form to prevent action until needed

enzyme composition

apoenzyme= protein, inactive without cofactor

cofactor= allows enzyme to function, loose bind to tight (covalent/prosthetic group)

holoenzyme (catalytically active unit)= apoenzyme + cofactor

inorganic activator (ZN alkakine phosphatase) organic coenzyme (NAD+)

allosteric vs active site

all- where regulatory molecules bind (back)

act- where substraye binds

oxidoreductases

oxidation-reduction reaction between 2 substrates

transferases

the transfer of a group other than hydrogen between 2 substrates

hydrolases

hydrolytic cleavage of substrates

lyase

removal of groups from substrates without hydrolysis, products contain db

isomerse

interconversion of geometric, optical, or positional isomers

ligase

the joining of 2 substates molecules, coupled with breaking of the pyro-phosphate bond in adenosine triphosphate ATP or a similar compound

inhibitors

decrease rate of reaction, reversible or permanent

competitive inhibitor

similar to substrate, competes for active site

noncomp inhibitor

binds to allosteric site (back), changes structure of enzyme so it can’t bind substrate

uncomp inhibitor

binds enzyme-substrate and prevents release of product

purpose of testing enzymes

enzymes are concentrated in specific tissues and organs, “normal” enzyme concentration in plasma = normal cell turnover, increase= increases cell injury or synthesis, decreased= decreased synthesis or inherited deficiency

enzyme activity affected by temp and pH

=proteins, optimal within physiological range (37C, low slow, high denaturation 40-50), extreme pH= denaturation, controlled by incubation water baths/ buffers

enzyme activity affected by cofactors and inhibitors

= gas and brakes pedals, necessary cofactors must be in reagent, comp inhibitor can be out competed by increasing sub concentration

enzyme activity affected by substrate concentration

= direct relationship until saturation, low concentration(slow), high (faster), reagents include 10-100x excess substrate to ensure we reach saturation

enzyme activity affected by enzyme concentration

=target analyte, with all other variables controlled, enzyme activity is directly proportional to enzyme concentration

enzyme activity affected by temp, pH, cofactor, inhibitor, substrate concentration

= controlled by analzyer/reagent

michaelis-menten curve

relationship between substrate concentration and rxn velocity

maximum velocity- occurs at enzyme saturation, excessive substrate

Km= constant, [S] at ½ Vmax, unique to E with specific S, E-S affinity (lower affin higher Km)

V= Vmax [S] / Km + [S]

first order kinetics

velocity directly proportional to the substrate concentration

zero order kinetics

velocity plateau, velocity is independent of substrate concentration , linear, enzymes cannot work any faster, producing constant amount of product

enzyme measured by

change in absorbance (product formation, substrate depletion, coenzyme depletion, altered coenzyme formation)

enzyme measurement methods

fixed-time / discontinuous / end-point assay (rxn stopped at a specific time with addition of weak acid)

continuous monitoring / kinetic assay (more common)

enzyme activity unit

amount of enzyme that produces 1 umol of product per minute under standardized conditions, IU/L or U/L

direct enzyme measurement

electrophoresis or immunoassay for isoenzymes and isoforms

oxidoreductases

catalyze oxidation-reduction reactions between 2 substrates (lactate dehydrogenase)

transferases

catalyze the transfer of a group other than hydrogen between 2 substrates (creatine kinase)

hydrolases

catalyze hydrolytic cleavage of substrate (lipase and amylase)

tissue specificity

high- found predominately in one tissue, moderate- widely distributed in the body, low- found in most body tissue

lactate dehydrogenase LD LDH manifestations

myocardial infarction, hepatic disorder, hemolysis, carcinoma

lactate dehydrogenase LD LDH

oxidoreductase involved in anaerobic glycolysis, found in most cells of the body, highest amount in liver, heart, skel muscle, rbc

lactate dehydrogenase LD LDH significance

total increase or relative increase of one isoenzyme, cardio elevated in MI, myocarditis, shock, CHF, highest in pericious anemia, megaloblastic anemia

lactate dehydrogenase LD LDH measurement

forward, lactate to pyruvate yielding NADH, absorbance at 340nm

lactate + NAD with lactase dehydrogenase to Pyruvate + NADH + H

ref range 100-224 U/L

creatine kinase manifestation

myocardial infraction, skeletal muscle disorder

creatine kinase

transferase involved in ATP regeneration in muscle cells, stores phosphate as creatine phosphate for energy, highest amount found in skel muscle brain heart

creatine kinase significance

isoenzymes used in diagnosis of specific conditions, dimer with 2 subunits (m= muscle, b= brain), 3 combinations (CK-MM skel and card muscle, CK-BB brain and CNS, CK-MB cardiac muscle)

creatine kinase measurement

reverse reaction, hexokiinase- G6PD-NADPH system, increased 340 nm absorbance as NADPH is formed

creatine phosphate + ADP with CK to creatine + ATP then ATP + glucose with KH to ADP + G6PD then G6PD + NADP with G6PD to 6-phosphoglucanate + NADPH

CK ref range

male 46-171, female 34-145

CK-MB: less that 5% total CK

aspartate aminotransferase ALT or SGOT manifestation

myocardial infection, hepatic disorder, skeletal muscle disorder

aspartate aminotransferase ALT or SGOT

transferase (transfer of an amino group between aspartate and alpha keto acids), synthesis and degradation of amino acids, highest amount in liver (highest in acute hepatocellular disorders), heart, skel musc, kidney

aspartate aminotransferase ALT or SGOT measurement

decrease in absorbance at 340 nm

L-Aspartate + a-ketoglutarate <asorartate aminotransferase (P5P)> oxaloacetate + L-Glutamate

Oxaloacetate + NADH + H with malate dehydrogenase to malate + NAD

aspartate aminotransferase ALT or SGOT ref range

5-35 U/L

alanine aminotransferase ALT (AGPT) manifestation

hepatic disorder

alanine aminotransferase ALT (AGPT)

catalyzes transfer of amino group from alanine to a-ketoglutarate, highest amount in liver (more specific than ast, highest in acute viral or toxic hep, moderately increased in obstructive liver disease, hepatic cancer and cirrhosis)

alanine aminotransferase ALT (AGPT) measurement

decrease in absorbance at 340 nm

alanine + a-ketoglutarate with alanine aminotransferase (P5P) to pyruvate + L-Glutamate

Pyruvate + NADH with lactase dehydrogenase to L-Lactate + NAD+

alanine aminotransferase ALT (AGPT) ref range

7-45

alkaline phosphatase ALP manifestation

hepatic disorder, bone disorder

alkaline phosphatase ALP

frees inorganic phosphate from organic phosphate yielding alc, found in most tissue, highest amount in bone (osteoblast involvment= higher ALP) and LIVER

alkaline phosphatase ALP measurement

increase in absorbance 405 nm

alkaline phosphatase ALP ref range

4-15y= 54-369

males 20-50y= 53-128

males over 60y= 56-119

females 20-50y= 42-98

females over 60y= 53-141

gamma-glutamyltransferase GGT manifestation

hepatic disorder

gamma-glutamyltransferase GGT

transferase involved in pro synthesis, regulation of tissue glutathione, and transport of amino acids across membranes

found in liver, brain, prostate, pancreas, kidney

elevated in alcoholism, hepatobiliary disorders, pancreatitis, diabetes

gamma-glutamyltransferase GGT measurement

increased absorbance at 405 nm

Y-Glutamyl-P-nitroanilide + Glycylglycine to P-nitroaniline (yellow) + Glutamylglycylglycine

gamma-glutamyltransferase GGT ref range

male 6-55

female 5-38

amylase AMY manifestation

acute pancreatitis

amylase AMY

hydrolase involved in the digestion of starch, secreted by salivary glands and pancreas, diagnose pancreatitis, elevated in gastric and duodenal uulcers, renal disease, narcotic use, mumps

amylase AMY measurement

synthetic carb hydrolyzed by amylase producing color change measured spectrophotometrically

amylase AMY ref range

30-220

lipase LPS manifestation

acute pancreatitis

lipase LPS

catalyzes the hydrolysis of triglycerides, primarily sourced from pancreas, elevated in acute pancreatitis, pancreatic carcinoma, kidney disease, duodenal ulcers, intestinal obstruction

lipase LPS measurement

synthetic lipid substrate measuring the rate of color formation spectrophotometrically

lipase LPS ref range

under 38

electrolytes

ions capable of carrying an electrical charge, needed for fluid balance, nerve conduction, muscle contraction, acid base balance, enzyme function

water

60% of body weight, intracellular fluid ICF = 2/3, extracellular fluid ECF= 1/3 (intravascular- plasma, interstitial- surrounding cells, transcellular- body fluids like csf synovial pleural peritoneal)

electrolyte distribution is maintained by

diffusion and active transport

Na/K ATPase pump: ATP pumps 3Na out f the cell and 2 K in

intracellular and extracellular lytes

i- K, Mg, phosphate

e- Na, Cl, HCO3

osmolality

concentration of solute(milliosmoles) in a solution

increase affect on hypothalamus- increase in thirst, arginine vasopressin hormone AVP/ antidiuretic hormone ADH secretion by the posterior pituitary then reabsorb by the water

decrease causes opposite

osmolality measured by

freezing point depression (more dissolved particles= lower freezing point), vapor pressure decrease

Na and accompanying anions 90% and glucose urea and other 10%

osmolality ref range

serum 275-295 mOm/kg

urine 1-3

random urine 50-1200

osmolal gap

increased with ethanol, methanol, and ethene glycol

=measured- calculated osmolality

ref range 5-10

calculated osmolality

RAAS (renin angiotensin aldosterone system)

adequate blood volume= good bp= good perfusion, vasoconstriction, control Na and H2O

RAAS process

low blood volume and pressure detected in kidney, renin secreted by juxtaglomerular cells, converting liver angiotensinogen to angiotensin I, lung angiotensin converting enzyme ACE becomes angiotensin I and II, angiotensin II simulates vasoconstriction and secretion of aldosterone from adrenal gland, increasing reabsorption of Na (water follows) and secretion of excess K

atrial natriuretic peptide ANP and B-type (brain) natriuretic peptide BNP

released from heart atria due to volume expansion or stretching, opposes RAAS (promotes Na and H2O excretion), hypervolemia (congestive heart failure CHF)

sodium Na

major extracellular cation, water balance, and blood volume and pressure regulation, hypoNAtremia (diuretics, prolonged vomit/diarrhea), hyperNAtremia (dehydration), ISE

Na ref

serum plasma 136-145

urine 120-240

csf 136-150

Potassium K

major intracellular cation, nerve conduction, muscle contraction, cardiac rhythm, hypoKalemia (diuretics, prolonged vomit/dia, insuline administration), hyperKalemia (renal failure, psuedohyperkalemia- hemolysis), ISE

K ref

3.5-5.1

chloride Cl

major extracellular anion, water balance, electroneutrality (follows Na), acid base balance, hypoCHLORemia (prolonged v/d, metabolic alkalosis), hyperCHLORemia (dehydration, metabolic acidosis), ISE

Cl ref

98-107

bicarbonate HCO3

major extracel anion, acid base balance (HCO3 and Co2 work together, HCO3 is base accepting H), decrease= metabolic acidosis, increase= alkalosis

bicarbonate HCO3 measurment

reported as total CO2 (plasma CO2 mainly exists as HCO3)

coupled enzymatic rxn (decrease in NADH absor)

CO2 + H2Oto H2CO3 to H+HCO3

HCO3 ref

22-33

calcium Ca2

extra cat, 99% in bone 1% in blood and fluid, muscle contraction, nerve transmission, coagulation, hypoCALCemia (hypoparathyroidism low PTH, vit D deficiency), hyperCALCemia (hyperparathyroidism high PTH)

Ca2 regulated by

parathyroid hormone PTH, vit D, calcitonin

Ca2 measurment

total calcium- colormetric assay, ionized 50%, protein(alb) bound 40%, small ion(phosphate, bicarb) bound 10%, may be decreased by low alb

ionized calcium- ISE, physiologically active form, unaffected by alb

Ca2 ref

total calcium serum plasma- kid 2.13-2.63, adult 2.24-2.53

ionized Ca serum- 1.15-1.33 whole blood- 1.15-1.27

Magnesium Mg2

intra cat, enzyme cofactor, ATP metabolism, neuromuscular func, hypoMAGNESemia (low K and Ca2, alcoholism, malnutrition), hyperMAGNESemia (renal failure)

Mg2 measurement and ref

colormetric, xylidyl blue

serum 0.66-1.07

phosphate

major intra anion, component of ATP, bone mineralization, acid base buffering, hypoPHOSPHATemia (malnutrition, hyperparathyroidism), hyperPHOSPHATemia (renal failure, hypoparathyroidism)

phosphate measurement and ref

colorimetric ammonium molybdate

serum adult 0.81-1.45

lactate

byproduct of anaerobic metabolism, component of ATP, bone mineralization, acid base buffering, increased lactate (tissue hypoxia, poor perfusion, shock)

lactate measurement

couples enzymatic lactate oxidase peroxidase

no tourniquet/fist pumping, heparin or sodium fluoride, deliver to lab on wet ie separate ASAP

lactate ref

plasma venous- 0.3-2