418 chem exam 1

gold standard for inborn error of metabolism

Gold standard: HPLC tandem mass spectrophotometry, can use dried blood spots

phenylketonuria

phenylalanine hydroxylase defect, body cannot convert phe to tyrosine
treat with low protein diet/limit phenylalanine

alkaptonuria

buildup of homogentisic acid from converting phe to tyr, black pee
treat with low protein diet and nitisinone to block enzyme

maple syrup urine disease

body can’t break down branched chain aas, toxic maple syrup odor in urine
treat with low protein diet or liver transplant

plasma vs serum

plasma contains fibrinogen and is treated with anti-coagulants

serum is devoid of fibrinogen and is clotted

measuring total protein

spectrophotometric biuret, copper in alkaline solution

measure albumin

BCG or BCP

albumin measurement interferences

low result for penicillin and anion drugs that bind to albumin at same site as BCG

immunoassay for specific proteins

turbidimetry and or nephelometry, or ELISA

turbidimetry vs nephelometry

turb measures reduction of light, neph measures scattered light at 90 degrees

electrophoresis

can use to measure specific proteins
cathode is negative charge, anode is positive charge

polyclonal immunoglobullins

indicative of inflammation, infection, liver disease

monoclonal immunoglobulins

B lymphs produce homogenous, indicative of multiple myeloma, leukemia, walden stroms macroglobinemia(IgM)

order of major proteins

albumin, a1, a2, b, y

prealbumin(transthyretin)

marker for protein nutrition status, measured by turb/neph, transports thryoxine and retinol, negative acute reactant

albumin reference range

3.5-5.2 g/dL

albumin

significant contributor to oncotic/osmotic balance, marker for nutrition, liver capacity, kidney or GI disease, binds variety of ligands, may affect interpretation of ca and mg concentrations, negative acute reactant

microalbumin(urine albumin)

diabetic monitoring, measure by turb/neph or dipstick, 24hr collection preferred

a1 anti trypsin

serine protease inhibitor that is synthesized in the liver, causes issues to lungs and or liver if deficient, positive acute reactant

haptoglobin

a2 globulin, scavenges hemoglobin, synthesized in liver, acute phase reactant

ceruloplasmin

a2 glubulin, copper carrying protein

wilson’s disease

copper excess, defect in transporter
low serum ceruloplasmin, high liver copper, high urine copper

menke’s

copper deficiency
low serum ceruloplasmin, low liver copper, high urine copper

transferrin

beta globulin transports iron, negative acute reactant

ferritin

beta globulin acts as iron storage, positive acute reactant

IDA vs hemochromatosis

IDA: increased transferrin, decreased ferritin
hemochromatosis: increased ferritin(iron overload)

b2 transferrin

CSF specific transferrin, detect by immunofixation

b2 microglobulin

low serum levels and high urine levels in renal tubular disease, increased in kidney heavy metal damage, aid in prognosis of multiple myeloma

y globulin CRP

has ability to bind c polysaccharide on s.pneumoniae(>10 mg/L may be infxn), high levels may be prognostic for cardiovascular risk
hsCRP more sensitive alternative

creatine kinase/creatine phosphokinase

atp generation in contractile or transport system, catalyzes reversible reaction of creatine to phosphocreatine

ck tissue source

highest in skeletal muscle, heart, brain
serym concentration positively correlated with muscle mass

ck conditions

elevation associated with disorders of cardiac muscle
skeletal muscle: rhabdomyolysis/crush syndrom
duchenne type muscular dystrophy, alcoholic myopathy

CK assay

1: reaction ck specific
2 and 3: coupled reactions with hexokinase and g6pd
nadh appearance measured at 340nm

ck assay errors

hemolysis may elevate ck activity, ck inactivated by light

ck isoenzymes

dimers of two subunits
ck mm: 94% of total ck in serum
ck bb: in brain, low concentration in serum, very high levels associated with death
ck mb: heart marker, low activity in serum

LDH

catalyzes interconversion of lactate and pyruvate, tissue and disease non specific marker of cellular and tissue inflammation and damage
2 subunits M(muscle) and H(heart), five isoenzyme forms rarely measured

LDH tissue source

heart, liver, skeletal muscle, rbcs
if elevated, other tests helps determine which organs involved

AST/SGOT

aspartate aminotransferase, significant indicator of liver damage

AST significance

hepatocellular disorders, skeletal muscle disorders, pulmonary embolism
large elevation: acute/toxic hepatitis
moderate elevation: cirrhosis
obstructive jaundice if ALT also norm/slightly high and ALP and GGT more elevated

AST measurement

Karmen method: oxaloacetate and malate dehydrogenase
measure NAHD at 340nm

AST assay concerns

hemolysis

stable in serum for 3-4 days refrigerated

ALT/SGPT

alanine aminotransferase, more specific to liver than AST, significant for hepatic disorders

ALT tissue source

high concentration in liver, other sources

AST/ALT ratio

normal less than or equal to 1, elevation ratio related to poor prognosis
>3: alcoholic hepatitis and cirrhosis

ALT assay

coupled enzymatic reaction using LDH as indicator enzyme, measure NADH at 340nm

ALT assay concerns

relatively unaffected by hemolysis, stable for 3-4 days at 4C

ALP

generic name for group of hydrolases that catalyze cleavage of phosphate monoesters in alkaline pH, requires Mg cofactor

ALP tissue source

mostly liver and bone, spleen and placenta

serum ALP bone

bone disease associated with increased osteoblastic activity

serum ALP hepatobiliary

increased synthesis due to impaired excretion due to biliary tract damage or obstruction, post hepatic jaundice
2-3x in intrahepatic obstruction
10x in extrahepatic obstruction

ALP assay

ELP for total or isoenzymes, colorless to yellow at 405nm

ALP assay errors

hemolysis, high fat meal
assay run ASAP after collection

GGT

elevated in ALL forms of liver disease: assess biliary epithelial damage and obstruction, determine whether other liver enzyme elevations are due to skeletal disease or other hepatic sources
acute pancreatitis, diabetes mellitus, myocardial infarction

GGT tissue source

hepatobiliary tract, kidney, brain, prostate, pancreas

5’ nucleotidase

same clinical utility as GGT

GGT assay

the dye p-nitroaniline released

GGT assay concerns

hemolysis not a concern, stable for 1 week at 4C

G6PD

deficiencies are sex linked or clinically manifested in drug induced hemolytic anemia, myocardial infarction, megaloblastic anemias
generates NADH, reduces oxidant burden

G6PD tissue source

adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland, rbcs

G6PD assay

quantitative red cell hemolysate to assay for deficiency at UW NW campus

macroenzymes

high molecular mass forms of the serum enzymes bound to immunoglobulin(type 1) or a non immunoglobulin substance(type 2), rare but in unexplained persistent increase of enzyme activities in serum
ID by PEG, protein electro, etc

measure enzyme concentration

mass of enzyme via immunoassay or other sensitive assay, less commonly done

measure enzyme activity

rate of substrate conversion to product or rate of product formation

endpoint versus kinetic

endpoint: Af - Ai
kinetic: multiple measurements at specific time intervals

U/IU

amount of enzyme that catalyzes the reaction of 1 umol substrate/min under the specific conditions of the assay

inorganic cofactor

non protein chemical compound that binds to an enzyme and is required for activity, activator

coenzyme

an organic cofactor

holoenzyme/apoenzyme

enzyme without its cofactors

6 common enzymes

oxidoreductase
transferases: transfer C, N, or P containing groups
hydrolases: cleavage of bonds by addition of water
lyases: cleave cc, cs, and some cn bonds
isomerases: racemization of optical or geometric isomers
ligases: formation of bonds between c and o,s,n coupled to hydrolysis of high energy phosphates

vmax

reaction rate when enzyme is fully saturated by substrate, all binding sites are constantly reoccupied

Km

substrate concentration when half of the active sites are occupied, equal to concentration of substrate at half vmax

zero order kinetics

excess substrate: constant amount of substrate converted per unit time

first order kinetics

excess enzyme: amount of substrate converted per unit of time is proportional to concentration, constant % per unit time

competitive inhibitor

binds to active site: km increases, vmax unchanged

non competitive inhibitor

binds allosterically, slowing down the enzyme: km unchanged, vmax decreases

uncompetitive/mixed inhibitor

binds only to the enzyme substrate complex: km decreases, vmax decreases

ketose vs aldose

ketose: C double bond to O, single to two Cs
aldose: C double bond to O, single to C and H

monosaccharide

4-6 carbons: glucose, galactose, fructose

disaccharide

2 monosaccharides: maltose, lactose, sucrose

polysaccharide

starch, glycogen

digestion

1: salivary amylase
2: pancreatic amylase hydrolyzes starch to disaccharides in duodenum
3: disaccharides broken down further by enzymes at microvilli of intestine
4: absorption of glucose and other monos, transport to liver via hepatic portal venous supply

glycolysis

aerobic: generates pyruvate ATP, NADH via CAC, and oxidative phosphorylation
hexose monophosphate shunt

glycogenolysis

short fasts, blood glucose maintained from glycogen in liver

gluconeogenesis

long fasts, glycogen stores exhausted: glucose regenerated from non carb sources

endocrine regulation

insulin only hormone that lowers serum glucose

hypoglycemia ranges

low: <70 mg/dL
critical <50 mg/dL

hyperglycemia

increase in plasma glucose concentration caused by an imbalance of hormones related to increased fasting serum glucose, symptoms if >200 mg/dL
associated with diabetes mellitus, liver failure, polyuria and polydipsia

glucosuria

renal threshold for glucose: plasma glucose >160-190 mg/dL

fasting plasma glucose test

stand alone test, no food or drink 8-12hrs before, component of oral gllucose tolerance test
normal: 70-99
provisional diabetes diagnosis: greater than or equal to 126

ADA diabetes mellitus criteria

1: HbA1c greater than or equal 6.5%
2: symptomatic and random glucose level greater than or equal 200 mg/dL
3: fasting plasma glucose greater than or equal 126 mg/dL

4: oral glucose tolerance test greater than or equal 200 mg/dL two hours after 75g glucose load

diabetes testing for adults >45 years

fasting glucose every 3 years, more frequent if risk factors

pediatric diabetes testing

retest every 2 years: risk factors, symptomatic at 10 or onset of puberty

insulin resistance

waistline >40in(men), >35in(woman), bp over 130/80, fasting glucose >100 mg/dL, fasting triglycerides >150 mg/dL, HDL >40(male) >50(female), skin tags, acanthosis nigricans

type 1 diabetes mellitus

damaged pancreas makes decreased or no insulin, related to destruction of beta cells, develops in children/teens, excess glucagon, >1 autoimmune markers, greater tendency to produce ketones

type 2 diabetes mellitus

hyperglycemia due to insulin insensitivity or resistance, greater tendency to develop coma without ketoacidosis

gestational diabetes mellitus

placental estrogen, cortisol, and human placental lactogen can block mother’s insulin causing glucose elevated in blood with altered metabolism
screen 24-28wks, treat with diet/exercise or insulin

glucose challenge test

24-28wks, fasting not required:
drink with 50g glucose, measure serum glucose within 1hr, follow up with oral glucose tolerance test if abn
normal <140, abn >140

oral glucose tolerance test

patient fasting 8-14hrs, take baseline plasma glucose test, administer 75g anhydrous glucose in 250-300ml water, take prandial glucoseL
normal <140, provisional diabetes 200 mg/dL or higher

serum/plasma for glucose

lime green, orange, hold, red, gray
separate after clot formed, centrifuge within 30min
stableish with refrigeration: glucose decreases 10 mg/dL every hour at room temp