INSULIN Flashcards
(27 cards)
HbA1c monitoring uses immunoassay?
yes
what is glycated?
is hemoglobin A1c glycated?
glycated means there is addition of sugar molecules that are non-enzymatically added.
yes.
is glycated and glycosylation the same?
no.
glycated: non-enzymatic
glycosylation: enzymatic
what is the reaction that took place between glucose and amino end of the beta-chain in Hb?
condensation reaction
the higher the blood glucose level, the higher the levels of ____
glycated Hb (HbA1c)
the longer the periods of _____, the higher the levels of ____
high blood glucose levels; glycated Hb (HbA1c)
why is HbA1c levels used for long-term monitoring of patient’s diabetic condition?
because HbA1c remains throughout RBC’s lifespan
what is the method of immunoassay used for DCA 2000 HbA1c kit?
inhibition of agglutination
principles of DCA 2000 HbA1c
- agglutinator is added into the kit
- synthetic polymer containing multiple copies of immunoreactive portion of HbA1c - antibody is added into the kit
- antibody is anti-HbA1c mouse monoclonal antibody derived from mouse, coated onto latex particle - patient blood sample added
- if there is sufficient HbA1c in the blood, they bind competitively to the antibody, preventing the agglutinator from binding. no agglutination. –> low scattering, decreased absorbance
is blood glucose monitoring using glucose test strips and glucometer a type of immunoassay?
no. does not make sense to have antibody targeting glucose
what is insulin?
a hormone that regulates blood glucose by promoting shift of glucose form circulatory compartment to intracellular compartment
process of making insulin in vivo
- beta cells in islets of langerhans of pancreas produce pre-proinsulin that contain a 23 aa amino-terminal signal sequence (signal peptide)
–> is infront of beta chain - cleave the signal peptide (23 aa amino-terminal signal sequence) –> form proinsulin
- trypsin and carboxypeptidase cleave C peptide –> form insulin
mature insulin consists of chain A (21 aa) and chain B (30aa) joined by 2 disulfide bonds
difference between human insulin and bovine insulin
3 amino acid variations at A8, A10, B30
difference between human insulin and porcine insulin
a single amino acid variation at B30
why is the difference between human insulin and bovine + porcine insulin a concern?
slight difference can lead to immune response (lipoatrophy, local allergic reaction –> itchy, redness)
what happens if proinsulin is not totally removed during purification?
lipoatrophy and local allergic reaction
steps of producing of recombinant human insulin using proinsulin method
- proinsulin mRNA undergoes reverse transcription to form proinsulin cDNA
- chemically synthesised methionine (Met) codon (ATG start codon) is attached to 5’ end of proinsulin cDNA
- vector is inserted (?) into the cDNA and then it is inserted into a host cell
- chemical cleavage by cyanogen bromide (CnBr) to remove Met residue from proinsulin polypeptide chain
- proinsulin chain is subjected to folding process to allow intermolecular disulfide bond formation
- C-peptide cleaved by enzymes (trypsin and carboxypeptidase) to yield human insulin
properties of insulin (3)
- likes to form dimers and higher order associated states due to favourable hydrophobic interactions –> does not exist as monomer –> precipitates
(monomers are the ones binding to the insulin receptor and hence they do not exert their action immediately) - in the presence of zinc, insulin is also able to form zinc-containing hexamer
- hexamer dissociates into monomer at site of injection when injected concentration falls to physiological levels
types of insulin
- rapid-acting (insulin lispro, -aspart, -glulisine)
- intermediate-acting (NPH = neutral protamine hagedorn insulin)
- fast acting (insulin glargine, -detemir)
how do rapid-acting insulin work
all three analogues have modifications on the beta chain –> they like to form monomers –> exert effects fast
modifications of rapid-acting insulin
lispro = Humalog: LysB28 and ProB29 reversed, to resemble insulin-like growth factor (IGF-1) that poorly self associates –> exist as monomer
aspart = Fiasp, NovoLog: ProB28 changed to AspB28 –> reduction in association –> non-hexameric
glulisin = Apridra: LysB28 changed to GluB28 and AsnB3 changed to LysB3 –> reduction in association –> non-hexameric
properties of intermediate-acting insulin (3)
NPH (neutral protamine hagedorn) insulin = insulatard, humulin : aka isophane insulin, is a cloudy suspension
regular insulin and protamine co-crystallised with zinc in neutral pH using phosphate buffer. form neutral crystalline insulin suspension.
can be readily mixed with regular insulin (extemporaneously or in commercial products, 70/30, 50/50 NPH/regular) –> helps to cover post-meal hyperglycemia
how do intermediate acting insulin work
they hexamerise and crystallise with the addition of protamine, this causes the insulin to take awhile for it to dissociate into monomer
modifications of slow-acting insulin glargine + how it works
insulin glargine = lantus:
alpha chain - AsnA21 changed to GlyA21
beta chain - 2Arg residues added to C-terminus
these modifications lead to change of glargine’s isoelectric point to 6.7, which is higher than the isoelectric point of regular insulin at 5.4. the increase in isoelectric point makes glargine closer to physiological pH in body, and since protein’s solubility is lowest at its isoelectric point, glargine is the least soluble in body.
