17 Biochemistry Lab - Genetic Basis of Cancer PDF

WEEK 17  Resting/non-dividing stage (G0) Cells are quiescent and not actively dividing Cell are either temporarily or permanently in G0 SENESCENT CELL – a stage in which cells can no longer divide(permanently in G) QUIESCENT CELLS – cells that do not divide...

WEEK 17  Resting/non-dividing stage (G0) Cells are quiescent and not actively dividing Cell are either temporarily or permanently in G0 SENESCENT CELL – a stage in which cells can no longer divide(permanently in G) QUIESCENT CELLS – cells that do not divide when it reaches maturity or terminal differentiation(e.g cardiac muscle, nervous tissue) CELL REGENERATION CYCLE Actively dividing stage Assures that cell accurately duplicates its contents cOmposed of 4 phases G1/post mitotic phase -cells have normal amount of DNA(2N) and are in the process of preparing for DNA duplication; long gap(6-12Hours)- more prone to damage ; disrupted in cancer S/DNA synthesis phase - synthesis and duplication of DNA(4N) G2/pre mitotic phase - cells are being prepared for cell division M/Mitotic phase - Results in cell division and formation of 2 daughter cells.  Cell cycle regulation Process assures that cell accurately duplicates its contents Important checkpoints – G1 and G2 – regulated by protein kinases called CYCLINS(cdk) CHECKPOINT determine whether the cell proceeds to the next phase of the cycle. Checkpoints Determine whether the cell proceeds to the next phase of the cycle or not 2 important checkpoints are present at G1 and G2 and regulated by protein kinases CYCLINS(cdk) Restriction points Pause in response to ENVIRONMENTAL cues After passing this point, the cell is committed to completing the rest of the cycle and dividing Wait for molecular signals Regulatory signals Either stimulators (protooncogenesis) or inhibitors(tumor suppressor gene) of the cell growth Stimulatory gene product – hyperactive Inhibitory gene product- inactive Will lead to cancer formation  G1/S checkpoints Most often disrupted in CANCER Occurs in late G1 when cells must commit and complete DNA synthesis Affected by extracellular stimuli such as growth factors Activation of G1 cyclin-CDK complex by extracellular factors (e.g. growth factors) phosphorylate the Rb gene Phosphorylation of the Rb gene leads to activation of transcription factor E2F= cell cycle progression continues Rb (retinoblastoma)gene– a tumor suppressor gene which normally binds the E2F G2/M checkpoints Mechanism of regulation is complex and results in Determines whether the cell is prepared to proceed activation of several genes needed for s phase to mitosis progression NOT affected by EXTRACELLULAR stimuli promotes differentiation through association with Regulated by CYCLIN B/CDK1 or CDC2(mitosis transcription factors promoting factor/MPK E2F= family of transcription factors in eukaryotes MPK is activated by the success of previous processes Activity of this with its substrate results in chromosomal condensation nuclear membrane breakdown spindle formation Protooncogenes ❑ "Proto" means the pre-form of oncogene ❑ They are not cancerous genes; they are your healthy genes ❑ They are also the one that operates your cell cycle ❑ Proteins that signal the cell cycle to go on ❑ Gene transcription → translation → protein ❑ Occur naturally ❑ Normally activated when increased cellular proliferation is required, i.e. embryonic development ❑ Kept in check by tumor suppressor (TS) genes  Cancer cell become invasive through the following ▪ Alteration of the surface biochemical structure of cancer cells ▪ Increased motility of cancer cells ▪ Elaboration of lytic substances by cancer cells ▪ Decreased mutual adhesiveness of cancer cells ▪ Loss of mutual growth restraints between cancer cells and normal cells.4. Invasion and Metastasis Invasion ❑ Active translocation of neoplastic cells across tissue barriers ❑ Critical pathologic point: ❑ Local Invasion ❑ Neo-vascularization ❑ for cancer or a tumor cell to move to another site, it needs new blood vessels to make it successful ❑ Angiogenesis - formation of new blood vessels ❑ Tumour vessel number correlates positively with risk and degree of dissemination ❑ Several cytokines that stimulate endothelial cell proliferation also stimulate the proliferation of malignant cells. ❑ A cancer cell cannot survive without a new blood vessel because it supplies blood for oxygen and fuel for the cancer cells to grow. ❑ Glucose is the fuel for cancer cells ❑ In stressed people, the hormone cortisol is constantly Increased which promotes gluconeogenesis resulting in prolonged hyperglycaemic state that will allow the cancer cells to live ❑ This explains why stressed people are predisposed to develop cancer eventually. ❑ May occur before clinical detection Cancer Metabolism Cancer metabolism refers to the unique ways in which cancer cells generate energy and build the molecules they need to grow and spread. Unlike normal cells, cancer cells often rely on altered metabolic pathways to meet their increased energy demands and support rapid proliferation. Here are some key aspects of cancer metabolism: 1. Warburg Effect Description: Cancer cells preferentially use glycolysis to produce energy, even in the presence of oxygen, a phenomenon known as the Warburg effect. Mechanism: This process generates less ATP (energy) than oxidative phosphorylation but produces intermediates essential for cell growth and division. Impact: The Warburg effect allows cancer cells to thrive in low-oxygen environments and supports their rapid growth. Cancer Metabolism 2. Altered Glucose Metabolism Description: Cancer cells consume more glucose than normal cells. Mechanism: Increased glucose uptake and glycolysis provide the necessary building blocks for biosynthesis and energy production. Impact: Targeting glucose metabolism is a potential therapeutic strategy to starve cancer cells. Cancer Metabolism 3. Amino Acid Metabolism Description: Cancer cells have altered amino acid metabolism to support their growth. Mechanism: They rely on amino acids like glutamine for energy production and biosynthesis. Impact: Targeting amino acid metabolism can disrupt the growth and survival of cancer cells. Cancer Metabolism. 4. Lipid Metabolism Description: Cancer cells often have increased lipid synthesis and uptake. Mechanism: Lipids are essential for building cell membranes and signaling molecules. Impact: Inhibiting lipid metabolism can impair cancer cell growth and proliferation. 5. Mitochondrial Function Description: Cancer cells may have dysfunctional mitochondria, leading to altered energy production. Mechanism: They rely more on glycolysis and less on oxidative phosphorylation. Impact: Targeting mitochondrial function can be a strategy to selectively kill cancer cells  Classification of ONCOGENES POINT MUTATION Secreted growth factors c-sis, hst Single change Cell surface receptors erb-B, fms, ret, trk, Ras gene- encodes for p21 –a CDK (cyclin dependent kinase )inhibitor in the cell cycle Intracellular transducer c-src, c-abl, mst, ras promotes cell cycle arrest DNA – binding nuclear proteins myc, jun, fos Ras- GTPase protein Regulators of cell type bcl, bax, bad Norma Ras – binds to GTP in the cell membrane and has GTPase activity upon binding GTP-Ras – transmit the signal in the cell leading to The first four are part of the SIGNAL TRANSDUCTION transcription of p21 PATHWAY MECHANISMS OF ONCOGENE ACTIVATION There are 4 ways by which oncogenes are activated ❖ POINT MUTATION ❖ GENE AMPLIFICATION ❖ GENE TRANSLOCATION ❖ VIRAL INTEGRATION  In bladder cancer, the Ras proto-oncogene is converted to a Ras oncogene(H-Ras) by a single missense mutation Substitution of valine for glycine at codon 12 The mutation makes the mutated H-Ras lose its GTPase activity upon binding to GTP – NO transcription of p21–- (-) arrest in G1 – resulting to A resident oncogene may become amplified during perpetual cell division duplications resulting to insertions homogeneously staining regions (HSR)- large insertion double minutes – small insertions Around 40% of neurofibromatosis contain up to 200 copies of n-myc gene C-myc is amplified in some carcinomas (Burkitt's lymphoma) Neu is amplified in breast cancer Formation of homogeneous staining regions as a result of DNA amplification. Once a locus is amplified by several rounds of DNA amplification, tandem repeats can be excised to form double minute fragments. These fragments can integrate into the locus of another chromosome, forming a homogeneous staining region (HSR). Because this process is reversible, double minute fragments and HSRs are amplified regions of DNA that are interchangeable.  Viral integration Cellular oncogene activation through the insertion of viral oncogenes and promoter regions Induction of DSB by Viral promoter regions are tenfold stronger than their cellular counterparts leading to ROS/NOS/Viral protein deregulation of the gene expression where they are inserted. DNA damage induces DDR (DNA damage response), ATM/ATR and P53 get activated to repair the damage HPV oncogenes deactivate the normal function of DDR molecules and DNA damage failed to be recognized. E7 degrades claspins and attenuate DNA damage checkpoints, while E6 degrades p53 and base excision repair gets suppressed so that the genomic DNA remains unrepaired and cell cycle proceeds  TUMOR SUPPRESSOR (TS) GENE FAMILY ❑ Prevent neoplastic transformation Knudson’s Two –Hit hypothesis ❑ Recessive gene ❑ In order for a particular cell to become cancerous , ❑ Both copies of the normal diploid suppressor genes must both of the cell tumor suppressor gens must be undergo mutation to allow for malignant transformation MUTATED ❑ Normal function – inhibit or put to brakes on the cell growth ❑ First (inherited) mutation/hit is appoint mutation or and division cycle the breaks from the cell cycle some other small change confined to the TS gene ❑ Mutation – cause the cells to IGNORE the network of ❑ Second mutation involves loss of all or part of the inhibitory signals, removing chromosome ❑ increase rate of uncontrolled growth= cancer ❑ Possible mechanism ❑ They follow the KNUDSON’S TWO HIT HYPOTHESIS ▪ non dysfunction , mitotic recombination ▪ de novo interstitial deletion ❑ In each case, one allele is devoid of any marker close to the TS gene ❑ If the patient is HETEROZYGOUS for any marker, the tissue losses HETEROZYGOSITY and becomes HOMOZYGOUS or hemizygous ❑ LOSS of HETEROZYGOSITY (LOH) is the key indicator for the existence of TS genes that may have undergone mutations.  CLASSIFICATION RB1 ❑ Cell adhesion molecules ❑ Retinoblastoma ❖ APC(adenomatous polyposis coli) ❖ rare childhood malignancy ❖ DCC(deleted in colorectal cancer) involving the retina ❑ Regulators of cell cycle ❖ forms ; 1. hereditary(40%) the ❖ RB1(retinoblastoma associated protein 1) childs inherits one mutant gene at ❖ Tp53 (tumor protein 53) the RB1 gene on chromosome13q14 through the PRB MECHANISM germline In normal cells, pRB is inactivated by phosphorylation and activated by 2. malignant- somatic cell dephosphorylation mutation of the normal copy of RB1 Two to 4 hours before a ell enters the S phase of the cell cycle, the pRB gene occurs is phosphorylated 3. two different The phosphorylation releases the inhibition of E2F – allows the cell to mutations occur at both loci of proceed to S phase the RB1 Loss of function mutations of the RB1 gene loci– formation of defective ❑ RB1 gene product is expressed in many pRB which is unable to inactivate/phosphorylate E2F– continuous other tissues including osteoblast, cellular division and proliferation of tumor cells fibroblast ant the skin The product of MDM2 (murine double minute)oncogene binds and ❑ RB1 gene is entirely or largely deleted in inhibits pRB –favors cell cycle progression many retinoblastoma tumor Some viral oncoprotein (adenovirus E1A,SV40T antigen, HPV E7) can ❑ RB1 product(pRB) – nuclear protein that bind, sequester, and degrade pRB acts by binding and inactivating E2F F1: Mechanisms of RB1 inactivation during normal cell proliferation and tumorigenesis and their effect on transcription of cell cycle and apoptotic genesA. In G1 phase, the active hypophosphorylated form of RB1 binds to E2F transcription factors to repress the expression of cell cycle and apoptotic genes. In S phase, RB1 is inactivated by phosphorylation (P) and releases most E2Fs to induce transcription of cell cycle genes. A fraction of RB1-E2F1 complexes persist at the promoters of apoptotic genes, thus repressing their expression. B. In cancer, RB1 can be inactivated by either mutations or hyperphosphorylation. RB1 loss leads to de-repression of both cell cycle and apoptotic genes, whereas hyperphosphorylation causes de-repression only of cell cycle genes. Thus, in cells lacking RB1, tumorigenesis can occur only if survival pathways protect cells from RB1-loss induced apoptosis by limiting E2F1 proapoptotic activity or if a second alteration, such as the abrogation of the p53 proapoptotic pathway, occurs.  4. Tp53/p53 Nuclear p53 protein ❑ Guardian of the genome; inhibit cell growth and proliferation ❑ Transcription factor ❑ Inhibit unruly amplification and mutation of the DNA ❑ Activated through phosphorylation ❑ Stimulate apoptosis due to DNA mutations ❑ Allow the binding of tetramers to specific ❑ Located at chromosome 17p13.1 receptors genes placed downstream of the ❑ Function: induces DNA repair or apoptosis p21 ❑ Inhibited by HPV ❑ Normal p53 permits the synthesis of p21 ❑ Inactivated by point mutation p21 inhibits the CDK ❑ 80% missense mutations ---- amino acid substitution cells with damaged DNA are cannot ❑ Inactivated less frequently by deletion or by the action of pass G1 an inhibitor (MDMZ gene product E6 of HPV ) ❑ Mutations: point>deletion Mutator gene family ❑ Normal p53 protein can be inactivated by forming complexes ❑ First described in Escherichia coli(mutt-HSL- with cellular proteins or by proteolysis system) ❑ ex. HPV oncoproteins degrade p53 ❑ Checks for and corrects mismatched pairs ❑ Loss of p53 function occurs in ; ❑ Mutation of oncogenes and TS genes ❑ 70% of colorectal cancer ❑ directly involved in the cell cycle controls ❑ 50% of lung cancer which go wrong in cancer ❑ 40% of breast cancer ❑ Mutator genes have a general role in ensuring the ❑ It is the single most common genetic change in human neoplasia integrity of the genetic information ❑ Suppression of death by mutant p53 ❑ Mutations in mutator gene – inefficient replication ❑ stimulate inappropriate cell survival or repair of DNA ❑ stimulate cancer ❑ This leads to the formation of microsatellite instability (MIN)  Indirect carcinogens ❑ Stimulate metabolic conversion to produce ultimate carcinogen ❑ Metabolized by cytochrome p450 monooxygenase system ❑ e.g. polycystic aromatic hydrocarbon (PAHs), aromatic amines and azo dyes, aflatoxin B1 , nitrosamines and amides, asbestos, vinyl chloride,chromium, arsenic Direct carcinogens/proximate ❑ Inhibit chemical transformation or metabolism to be carcinogenic ❑ Highly electrophilic ❑ React with nucleophilic structures through non enzymatic reactions to produce covalent adducts ❑ Most are weak carcinogens ❑ e.g. alkylating agents like cyclophosphamide, chlorambucil, busulfan, melphalan, methylnitrosurea, and ethyl nitrosourea  CARCINOGENS AND ITS METABOLISM Carcinogenesis ❑ Process of tumor formation through the influence of different environmental agents ❑ Multi stage process that begins with a cell exposed to a potential carcinogenic agent resulting in damage to its INITIATION STAGE genetic apparatus ❑ Rapid, genotoxic (damages the genetic material ) ❑ Categories of carcinogens ❑ Irreversible ▪ occupational ❑ Result in the formation of mutant DNA ▪ lifestyle (tobacco, ❑ Initiation cells can still cleared by the body diet, etc ) PROMOTION STAGE immune system should exposure cease. ▪ multifactorial causes ❑ Formation of tumor ❑ However, if continued exposure occurs, the 2nd ▪ chemical cells phase of carcinogenesis occurs resulting in the carcinogens ❑ If untreated, will formation of a mass of tumor cells. ▪ biological result to metastatic disease and death  PHYSICAL UV rays ❑ Strong epidemiological relationship to squamous cell carcinoma, basal cell CARCINOGENS carcinoma, and melanoma ❑ Radial energy, whether in the ❑ Degree of risk absorbing protective dependent on type of UV rays, intensity of form of UV rays , ionizing exposure, and quality of light absorbing protective mantle of melanin in the skin radiation can transform virtually ❑ UV-B(280-320 nm)- responsible for the induction of cutaneous cancers all cell types in vitro and induce ❑ UV-C (200- 280 nm)- a mutagen but not considered significant, filtered out by neoplasm in vivo in man and ozone layers animals ❑ Causes formation of pyrimidine dimers in the DNA leading to a mutation – which ❑ Ionizing radiation have eventually repaired by nucleoside excision repair pathway. produced a variety of malignant ❑ With extensive exposure to UV light, the repair systems may be overwhelmed and melanoma skin cancer results ❑ Transmit irreversible changes in ❑ Individuals with defects in the enzymes that mediated DNA excision repair are gene expression to cell progeny especially susceptible ❑ Source of potentially ❑ Some effects of UV rays – inhibition of cell division, inactivation of enzymes, killing carcinogenic radiation of cells ❑ sunlight ❑ artificial sources of UV light ❑ x rays ❑ radio chemicals ❑ nuclear fission  IONIZING RADIATION ❑ Includes electromagnetic waves (x rays, gamma rays ) particulate (alpha, beta, protons, neutrons), and primarily cosmic radiation– therapeutic irradiation has been documented also to be carcinogenic ❑ All forms are carcinogenic with special sensitivity in ❑ bone marrow- acute leukemia occurs before other radiation-induced neoplasia ( 7 years latent period in atomic bomb survivors) ❑ thyroid gland – carcinoma occurs in 9% of those exposed during infancy or childhood. ❑ lung – increased frequency of lung cancer in miners exposed to radon gas. ❑ The oncogenic properties of ionizing radiation are related to its mutagenic effects ❑ causes chromosomal breakage translocations and less frequently, point mutation ❑ Double-stranded DNA breaks seem to be the most important form of DNA damage caused by radiation ❑ There is a hierarchy of vulnerability to radiation- induced cancers  Viral carcinogens ❑ Viruses are potential carcinogens ❑ The viral genome contains a strip of RNA or DNA that acts as an oncogene ❑ Viruses must stimulate the infected cell to proliferate for it to replicate itself ❑ This results in the incorporation of this oncogene into the genome of the host cells. DNA virus ❑ Normally infect cells lytically ❑ Cause tumors by rare anomalous integration into the DNA of nonpermissive host cells ❑ Viral transcriptional activation and replication signals are implanted into the host genome to trigger cell proliferation ❑ Some classes include ❑ T antigen of SV40 ❑ EIA and E18 of adenovirus ❑ These genes are virus specific and do not have cellular counterparts ❑ Transforming DNA viruses form stable association with the host cell genome ❑ integrated virus is unable to complete is replicative cycle because viral genes are interrupted during the integration of the viral DNA ❑ Viral genes that are transcribed early (early genes) in the viral life cycle are important for transformation ❑ they are expressed in transformed cells.  Human papilloma Virus (HPV) ❑ High risk or oncogenic HPVs are etiologic agents of cervical cancer ❑ Among the high-risk HPVs , HPV16 and HPV18 are the principal causes of cervical cancer as well as several other tumor types ❑ A characteristic of infection by these HPVs is that the viral genomes are commonly integrated into the cancer cells’ genome ❑ There is a constant site of interruption of viral DNA during the process of integration ❑ within E1/E2 open reading frame of the viral genome ❑ E1 normally represses transcription of E6, E7 ❑ E2 interruption causes overexpression of E6, E7 ❑ Two principal viral oncoproteins involved in cervical carcinogenesis are E6 and E7, which destabilize respectively, two cellular tumor suppressors, p53 and pRB ❑ E6 =p53 degradation ; E7= pRb ❑ Transmitted primarily through sexual contact /The best-studied of the tumor viruses ❑ HPV types ❑ squamous papilloma (warts)- 1,2,4,7 ; cervical/anogenital cancer – 31,33,51 ❑ The USFDA approved, the first cancer vaccine, for use in females 9-26 years of age to prevent cervical cancer ❑ Precancerous genital lesions, and genital warts caused by HPV6, HPV11, HPV16 and HPV18  EPSTEIN-BARR VIRUS (EBV) ❑ Primarily causes infectious mononucleosis ❑ Contributes to the pathogenesis of 4 human tumors ❑ the African form of Burkitt lymphoma ❑ b-cell lymphomas in immunocompromised individuals ❑ nasopharyngeal carcinoma (NPC) in Seasia ❑ some kinds of Hodgkins lymphoma ❑ EBV infects B lymphocytes but does not replicate within the B cells ❑ Instead, it transforms them into lymphoblasts, with an indefinite life span, rendering this cell immortal ❑ LMP1(latent membrane protein -1/BNLF1) ❑ LMP1 has expressed in EBV associated lymphomas and is essential for B-cell transformation and for disruption of cellular signal transduction ❑ prevents apoptosis by interacting with bcl-2 ❑ Although the EBV nuclear antigen 1(EBNA1) is one of the earliest viral proteins expressed after infection and is the only latent protein consistently expressed in viral associated tumors, recent results indicate that EBNA1 is not a viral oncoprotein ❑ EBNA1 transactivates several viral and host cell genes important for B cell growth ❑ BARF-1(BamH1-A reading frame -1) is also an early gene but is expressed as a latent gene in most NPCs  HEPATITIS B VIRUS ❑ Endemic in SEA and sun-Saharan Africa ❑ Associated with liver cancer ❑ the precise role of HBV is not clear ❑ viral genome does not code any oncoproteins ❑ no consistent pattern of integration in the vicinity of any known TS gene ❑ The effect may possibly be indirect or multifactorial ❑ HPV can cause chronic injury which expands the pools of cells at risk for subsequent genetic changes ❑ Produces HBx protein ❑ disrupts signal transduction ❑ deregulates cell growth through the inhibition of proteasome activities to enhance HBV replication in vivo ❑ HBx protein activates protein kinase C ❑ mimics the action of a tumor promoter ❑ Hbx also binds to and enhances the enzymatic activity of phosphatidylinositol3 kinase class III, an enzyme critical for the initiation of autophagy  RNA VIRUSES ❑ Have an RNA genome and replicate via DNA intermediate made by way of reverse transcriptase ❑ These viruses do not normally kill host cells ( except for HIV) and only rarely transform them ❑ Only one human retrovirus, the human T cell Leukemia Virus type 1(HTLV) is firmly implicated in the causation of cancer HUMAN T-CELL LEUKEMIA VIRUS (HTLV) ❑ The infection affects the expression of T-lymphocytes gene expression leading to increased proliferation of affected T-lymphocytes ❑ Primarily affects T-lymphocytes, specifically CD4+ ❑ HTLV-2 – predominantly affects CD8+ ❑ HTLV-1 – inhibit v-onc, and inhibit consistent integration next to a TS gene ❑ reveals the gag, pol, env, and Long terminal repeats ❑ Tax gene – a unique region on HTLV– products essential for viral replication ❑ stimulate transcription of viral mRNA by acting on 5’LTR ❑ activate transcription of several host gene cells ❑ c-fas, c-sis for IL-2 +receptor ❑ GM-CSF – myeloid growth factor ❑ Pathogenesis ❑ HTLV-1 stimulates the proliferation of T-cells through the action of tax protein, which turns on the genes encoding IL-2 and its receptor ❑ A paracrine pathway is activated by the increased production of GM-CSF that induces the macrophages to secrete IL-1 another T-cell mitogen ❑ The proliferative T cells are at an increased risk of secondary transforming events (mutations) which leads ultimately to the outgrowth of the monoclonal T cell population PRINCIPAL PATHWAYS FOR MALIGNANCY 1. STEPS/PATHWAY ❑ Occurs during the malignant transformation ❑ Main pathway involved ❑ Targets for development of anti cancer ❑ Receptor tyrosine kinase [RTK]pathway treatments ❑ Ras-MAP pathway – most prominent ❑ Pathways ❑ P13-kinase ❑ Proliferation ❑ Phospholipase ❑ cell cycle progression ❑ Ligand growth factors ❑ DNA repair ❑ NGF,PDGF,FGF,EGF ❑ immortalization ❑ Function ❑ Apoptosis ❑ promotion of cell survival ❑ angiogenesis ❑ regulation of cell proliferation and differentiation ❑ modulation of cellular metabolism CLINICAL IMPLICATION A.proliferation/growth factor signaling ❑ Inhibition of signaling pathway from the outside to the pathway onside ❑ uncontrolled due to the following factors ❑ prevents binding of growth factors to its receptors ❑ increased growth factors ❑ receptor dimerization with specific agents ❑ increased number of receptor ❑ Prevent receptor activation via small molecule inhibitors ❑ stimulate signal transduction ❑ Block downstream proteins of the activated receptor pathway  CELL CYCLE CDK inhibitors REGULATION/PROGRESSION ❑ Inhibit the activity of the CDK-cyclin complex ❑ cyclins and CDK are the important ❑ Two groups components of these steps ❑ INK4 family – p15,p16,p18,p19 ❑ checkpoint in G1 and G2 phase ❑ CIP-KIP family- p21, p27 ❑ determine whether the cell ❑ Actions proceeds to the next phase of the ❑ p15 – change response to antimitogenic agents cycle ❑ p16 – inhibit CDK4/cyclin D ❑ Cyclins and CDK is regulated by ❑ p19 – stimulate p53 stabilization CDK1[cyclin-dependent kinase ❑ p21 – stimulate cell cycle arrest via activation of p53 inhibitors… E.g.p15,p16,p18,p21] ❑ p21- associated with tumor suppressor CLINICAL SIGNIFICANCE ❑ Oncogenic alteration in cell cycle regulators ❑ p53- arrest the cell cycle at G1 ❑ Inhibit p53, pRB function as tumor suppressors ❑ CDK1 functions to inhibit the cyclin ❑ Increase expression of cyclin D1 (mantle cell lymphoma) complex and promotes the arrest of the ❑ CDK4 amplification in sarcoma, glioma cycle to make way for the cell damage ❑ Mutation in the p16-binding domain of CDK4 (familial repair mechanism before proceeding to melanoma) the next phase ❑ Inactivation of INK4 ❑ In cancer, checkpoints are disrupted so ❑ Alteration in cyclin D1,p16 the cycle progresses despite the ❑ Decrease p27 levels(breast cancer) presence of DNA mutations, thus ❑ Overexpression of cdc25 damaging cell proliferate  THERAPEUTIC IMPLICATION ❑ Inhibit CDKs via administration of small molecules, protein therapy, antisense oligonucleotides, gene therapy ❑ Most cytotoxic agents block the cell in the synthesis/G2/Mitosis ❑ still used now, but with targeted treatment utilizing the signal transduction process ❑ antimetabolite(blocks synthetic phase). 5- fluoroucil(5-FU), gemcitabine, methotrexate ❑ Anti-microtubule (blocks mitotic phase). Taxanes, anthracycline, vinca alkaloids ❑ Blocks G2 phase.. Bleomycin, etoposide  DNA REPAIR PATHWAY ❑ Cancer is a “malady of genes’ ❑ Gene mutation causes defects and repair mechanism ❑ Mechanism ❑ Mismatch excision repair – errors in replication ❑ Base excision repair – maintains DNA acting on spontaneous chemically induced damage ❑ nucleotide excision repair– UV and photoproduct damage ❑ Double strand base repair – errors due to ionizing and oxidative stress CLINICAL SIGNIFICANCE –HPNCC ❑ Hereditary non-polyposis colon cancer ❑ Autosomal dominant, highly penetrant, no preceeeding phase of polyposis, genes mapped at 2 codons- 2p21- 22 and 3p21.3 ❑ Involving somatic defects in DNA repair pathways leading to microsatellite instability (MSI)– MSH1, MSH2, inactivation of tumor suppressor TGF-beta and BAX gene  IMMORTALIZATION CLINICAL SIGNIFICANCE ❑ Telomeres are key elements in ❑ Most normal adult tissues have NO TELOMErase activity the immortalization of cancer ❑ In cancer cells, THERE is telomere activity in 90% of tumors cells ❑ Telomeres Therapeutic implications ❑ a specialized structure at ❑ hTERT- catalytic subunit of the telomerase chromosome ends ❑ Limiting component of the enzyme ❑ prevents loss of genetic ❑ Potential target for small molecular inhibitors materials ❑ generated and maintained by the enzyme telomerase ❑ Telomerase ❑ ribonucleoprotein which acts to preserve the integrity of telomeres ❑ Telomere length is like molecular clocks that determine the lifespan of a cell ❑ increase length telomers increase the lifespan  APOPTOSIS ❑ Programmed cell death. ❑ Importance ❑ Steady-state kinetics of normal tissues ❑ Focal deletion of cells during normal embryonic development APOPTOTIC PATHWAY ❑ Cell death is seen after chemotherapy and radiation ❑ FAS-mediated apoptosis ❑ The balance between proliferation and apoptosis is critical in determining growth ❑ FAS cell surface receptor or regression of TNF family which binds to FAS-L COMPONENTS OF APOPTOTIC PATHWAY ❑ Eliminates unwanted ❑ CASPASES activated T cells ❑ cysteine-containing aspartate specific proteases ❑ Pathway for cytotoxic – ❑ Initiator caspases – activated in response to cell death signal mediated signaling ❑ Executioner or effector caspases– progress the cell death signal activating ❑ P53 mediated apoptosis cascade resulting in DNA fragmentation and cell death ❑ Important after ❑ Caspases pro-domain– DED, CARD chemotherapy and ❑ Cytochrome c- component of mitochondria, released in response to apoptotic radiation signals ❑ Induction of BAX ❑ BCL-2 family of proteins – located upstream in the pathway ❑ Down- regulation of BCL- ❑ Provides pivotal decisional checkpoint in the fate of the cell after a death 2 stimulus ❑ Induced expression of ❑ Contain BH1-BH4 domains necessary for interaction FAS and DRS ❑ Anti-apoptotic- BCL-2, BCL-xl ❑ Pro-apoptotic – BAX,BAD,BAK,BID  CLINICAL SIGNIFICANCE ❑ overexpression of BCL-2 as a prognostic indicator ❑ mutation of BAX in gastrointestinal cancer and leukemia ❑ P53 mutation confers resistance to chemotherapy ❑ Cell survival signal – NFxB; BCL-2 Therapeutic implications ❑ Antisense oligonucleotide vs BCL-2 in the treatment of lymphoma ❑ BCL-2 antisense as a chemo-sensitizing agent in solid tumors ❑ TNF-related apoptosis- inducing ligand (TRAIL) to induce apoptosis  COMPONENTS OF ANGIOGENESIS ❑ Endothelial cell ANGIOGENESIS ❑ Fenestrated ❑ Formation of new ❑ Increase cell adhesion molecules (E-selectin) blood vessels ❑ Increase integrins – alpha gammabeta epsilon- essential for viability during growth from existing ❑ Activated EC release bFGF, PDGF,IGF-1 vascular bed ❑ Inducers of angiogenesis ❑ Carried out by ❑ VEGF (vascular endothelial growth factor)- main inducer endothelial cells ❑ TGF-beta; TNF-alpha(decrease concentration= inducer: increase concentration = inhibitor (EC) and ❑ PDGF/thymidine phosphorylase extracellular ❑ TGF-alpha: EGF;IL-8 (ECM) ❑ Cell adhesion molecules ❑ Regulated by ❑ Mediates cell to cell adhesion processes angiogenic factors ❑ Selectins (inducers and ❑ IG supergene family- iCAM, VCAM inhibitors) ❑ Cadherins ❑ A tum0r is unable ❑ Integrins – vitronectin receptor 4 to grow largen ❑ Proteases than 1 mm2 ❑ Degrade ECM to provide a suitable environment for EC migration through adjacent stroma e.e without metalloproteinases(MMP) developing a new ❑ Angiogenesis inhibitors blood supply ❑ Interferon; TSP-1; angiostatin; endostatin;vasosatatin  Clinical significance ❑ Tumor angiogenesis is triggered as a result of a shift in the balance of stimulators to inhibitors Therapeutic implications ❑ Metalloproteinase inhibitor (MMP1) – blocks the degradation of basement membrane ❑ Inhibitor of endothelial function ❑ Thalidomide, TNP 470,Endostatin ❑ Anti-angiogenic factors ❑ Tyrosine kinase inhibitors of VEGF, bFGF, PDGF ❑ Interferon – angiogenic inhibitor ❑ COX-2 inhibitor – thromboxaneA2 as critical intermedia of angiogenesis  INVASION AND METASTASIS PROTEOLYSIS ❑ Defining characteristics of malignancy ❑ Degradation of the basement membrane ❑ invasion- active translocation of neoplastic cells across tissue to transverse barriers barriers ❑ Carried by ❑ Clinical pathologic point ❑ Serine proteases – uPA; Elastase; ❑ Local invasion Plasmin; Cathepsin G ❑ Neovascularization ❑ Cysteine proteases – cathepsin B, ❑ These events may occur before clinical detection cathepsin L ❑ Aspartate proteases – cathepsin D TRIAD OF INVASION ❑ Matrix metalloproteinase – ❑ Adhesion with basement membrane gelatinase; intestinal collagenase; ❑ Local proteolysis stromelysins; matrilysin ❑ Motility MOTILITY ADHESION ❑ Tumor cells can move randomly or ❑ the deregulated function of CAM(E-cadherin) directionally towards attractants ❑ changes in catenin expression lead to loss of cadherin function ❑ Modulators of motility ❑ Integrin overexpression in naturally occurring cancers- ❑ GF, hyaluronidases; components of ❑ downregulation of integrin in more advanced stages of cancer ECM; tumor secreted factors; host- ❑ Upregulation of iCAM-1 which enhances extravasation derived factors ❑ Adhesion molecules on endothelial cells- E-selectin; VCAM; iCAM  THERAPEUTIC IMPLICATION ❑ MMPI ( matrix metalloproteinase inhibitors) and monoclonal antibodies against integrin LABORATORY ACTIVITY 18 BLOOD GLUCOSE DETERMINATION 1. Introduction Blood Glucose ❑ Blood glucose, also known as blood sugar, refers to the concentration of glucose present in the blood. ❑ Glucose is a crucial energy source for cells and is regulated within a narrow range in the bloodstream. ❑ Abnormal blood glucose levels can have significant clinical implications, particularly in conditions such as diabetes mellitus 2. Biochemistry ❑ Glucose is a simple sugar that serves as a primary source of energy for cells. ❑ It is derived from the digestion of carbohydrates in the diet and is released into the bloodstream where it can be utilized by cells for energy production. ❑ Insulin, a hormone produced by the pancreas, plays a key role in regulating blood glucose levels by promoting the uptake of glucose into cells and the storage of excess glucose in the liver and muscles. 3. Clinical Significance ❑ Maintaining blood glucose levels within a normal range is essential for proper cellular function. ❑ Abnormalities in blood glucose levels can indicate various medical conditions, including diabetes mellitus, hypoglycemia, and hyperglycemia. ❑ Diabetes mellitus is characterized by chronically elevated blood glucose levels due to insufficient insulin production or impaired insulin function. ❑ Hypoglycemia occurs when blood glucose levels drop below normal, leading to symptoms such as dizziness, confusion, and loss of consciousness. ❑ Hyperglycemia, on the other hand, is characterized by excessively high blood glucose levels and can lead to long-term complications such as cardiovascular disease, kidney damage, and nerve damage if left untreated. 4. Diagnostics ❑ Blood glucose levels can be measured using various diagnostic tests, including fasting blood glucose tests, oral glucose tolerance tests, and glycated hemoglobin (HbAc) tests. ❑ These tests help diagnose diabetes mellitus and monitor blood glucose control in individuals with diabetes. ❑ Objectives ❑ Obtain accurate measurements of blood glucose ❑ Reagents levels. ❑ Glucose mono-reagent: ❑ Aid in the diagnosis and monitoring of diabetes ❑ GOD 15ku/L mellitus. ❑ POD 1.0ku/L ❑ Acquire skills for determining glucose concentration ❑ Phenol 0.3mmol/L in a biological sample. ❑ 4-AP 2.6mmol/L ❑ Buffer pH7.55 92mmol/L ❑ Stabilizers and activators ❑ Equipment ❑ Glucose standard (glucose aqueous ❑ 1 mL measuring pipettes (2) primary standard 100 mg/dl) ❑ 5 mL measuring pipette (1) ❑ Cuvettes 1 cm light path (2) ❑ Micropipettes (2) ❑ Pipette Aspirator (1) ❑ Specimen for Examination ❑ Rubber stoppers (3) ❑ Serum or plasma ❑ Spectrophotometer (1) ❑ Test tube stand (1) ❑ Test tubes (5) ❑ Thermometer (1) ❑ Timer (1) ❑ Venipuncture set (1) ❑ Water bath (1) Procedures ❑ Label 3 test tubes as "blank," "standard," and "test." ❑ Add 1 mL of glucose mono-reagent to each test tube. ❑ Add 0.01 mL serum or plasma and standard to the appropriate test tubes. No addition to the "blank" test tube. ❑ Mix. ❑ Incubate at 37°C for 5 minutes or at 15-25°C (25°C) for 10 minutes. ❑ Set the spectrophotometer to "zero" using the blank test tube. ❑ Measure the absorbance (A) of both the samples and the standard at 505 nm using a spectrophotometer, comparing against the reagent blank. ❑ Calculation ❑ Glucose (mg/d1) = (A) Sample / (A) Standard * Standard Concentration  ❑ Laboratory Reports/Discussions ❑ What is the principle of the method used? ❑ Give and describe substances that may interfere with the test. ❑ Give the reference values. ❑ Give and describe conditions associated with elevated blood glucose levels. ❑ Self-Study Questions ❑ Basic Concepts ❑ What is glucose, and why is it essential for the human body? ❑ Describe the role of insulin in glucose metabolism. ❑ Explain the difference between hyperglycemia and hypoglycemia. ❑ Biochemistry of Glucose ❑ How is glucose synthesized in the body? ❑ What are the primary sources of glucose in the diet? ❑ Describe the process of glycolysis and its significance in glucose metabolism ❑ Regulation of Blood Glucose ❑ How does the body regulate blood glucose levels? ❑ Describe the roles of glucagon and insulin in blood glucose regulation. ❑ Explain what happens to blood glucose levels after a meal and during fasting. Here's why glucose is essential for the Brain Function Primary Fuel: The brain Metabolic Processes Glycogen Storage: human body: relies heavily on glucose Excess glucose is stored for energy. Unlike other in the liver and muscles tissues, the brain cannot Energy Production store glucose and as glycogen. This stored Cellular Respiration: Glucose is crucial depends on a constant glycogen can be for cellular respiration, a process that supply from the converted back to occurs in the mitochondria of cells. glucose when the body During cellular respiration, glucose is bloodstream. needs energy. broken down to produce adenosine Cognitive Function: triphosphate (ATP), the energy Fat Storage: When Adequate glucose levels currency of the cell. glycogen stores are full, are essential for Immediate Energy: Glucose provides a excess glucose is maintaining cognitive quick source of energy, especially converted into fat and functions such as important for brain function and stored in adipose tissue. physical activity. memory, attention, and learning.  5.Inhibition of Role of Insulin in Glucose Metabolism Gluconeogenesis: Liver: Insulin inhibits 1. Glucose Uptake: 2. Glycogen Synthesis: gluconeogenesis, the Muscle and Fat Cells: Glycogenesis: Insulin process by which the Insulin facilitates the stimulates the enzyme liver produces glucose uptake of glucose into glycogen synthase, 7. Inhibition of Lipolysis: from non-carbohydrate muscle and fat cells by which converts glucose Fat Breakdown: Insulin sources. This helps promoting the into glycogen for storage inhibits the breakdown prevent excessive translocation of glucose in the liver and muscles. of fats (lipolysis) in glucose production and transporter type 4 This helps maintain adipose tissue, reducing maintains blood glucose (GLUT4) to the cell blood glucose levels the release of free fatty levels. membrane. This allows within a normal range. acids into the glucose to enter the cells, bloodstream. This helps 6.Protein Synthesis: maintain energy balance where it can be used for Amino Acid Uptake: energy or stored as 3. Lipogenesis: and prevents excessive Insulin facilitates the fat breakdown. glycogen. Fat Storage: Insulin uptake of amino acids Liver Cells: In the liver, promotes the conversion into cells, promoting insulin promotes the of excess glucose into protein synthesis and uptake of glucose and its fatty acids and their muscle growth. This conversion to glycogen for storage as triglycerides anabolic effect is storage. This process is in adipose tissue. This essential for tissue known as glycogenesis. process is known as repair and growth. lipogenesis. Overall Effects Blood Glucose Regulation: By promoting glucose uptake, glycogen synthesis, and inhibiting gluconeogenesis, insulin helps maintain blood glucose levels within a normal range. Energy Storage: Insulin ensures that excess glucose is stored as glycogen or fat, providing a reserve of energy for future use. Anabolic Effects: Insulin supports the synthesis of proteins, fats, and glycogen, contributing to overall growth and energy storage. Explain the difference between hyperglycemia and hypoglycemia.​ Hyperglycemia vs. Hypoglycemia Hyperglycemia and hypoglycemia are conditions related to abnormal blood glucose levels, but they represent opposite extremes: Hyperglycemia Definition: Hyperglycemia refers to high blood glucose levels, typically above 180 mg/dL (10 mmol/L) after meals or above 125 mg/dL (7 mmol/L) when fasting. Causes: It can be caused by insufficient insulin production, insulin resistance, excessive carbohydrate intake, stress, illness, or certain medications. Symptoms: Common symptoms include frequent urination, increased thirst, blurred vision, fatigue, and headaches. If left untreated, it can lead to more severe complications such as diabetic ketoacidosis (DKA) or hyperosmolar hyperglycemic state (HHS). Management: Managing hyperglycemia involves monitoring blood glucose levels, adjusting insulin or medication doses, following a balanced diet, and regular physical activity. Hypoglycemia Definition: Hypoglycemia refers to low blood glucose levels, typically below 70 mg/dL (3.9 mmol/L). Causes: It can be caused by excessive insulin or medication doses, skipping meals, excessive alcohol consumption, or intense physical activity. Symptoms: Common symptoms include shakiness, sweating, confusion, irritability, dizziness, and in severe cases, loss of consciousness or seizures. Management: Managing hypoglycemia involves consuming fast-acting carbohydrates (e.g., glucose tablets, fruit juice), monitoring blood glucose levels, and adjusting insulin or medication doses as needed  Gluconeogenesis Process Key Enzymes: Several key 1. Substrates: enzymes are involved in Glucose Synthesis in the Gluconeogenesis uses gluconeogenesis: Body non-carbohydrate Pyruvate Carboxylase: Glucose is synthesized in substrates to produce Converts pyruvate to the body through a process glucose. These oxaloacetate in the called gluconeogenesis, substrates include: mitochondria. which primarily occurs in 1. Lactate: Produced by Phosphoenolpyruvate the liver and, to a lesser anaerobic glycolysis in Carboxykinase (PEPCK): extent, in the kidneys. This muscles and red blood Converts oxaloacetate to process is crucial for cells. phosphoenolpyruvate in the maintaining blood glucose 2. Glycerol: Derived from cytoplasm. levels, especially during the breakdown of Fructose-1,6-bisphosphatase: periods of fasting or triglycerides in adipose Converts fructose-1,6- intense exercise. Here's tissue. bisphosphate to fructose-6- how it works: 3. Amino Acids: phosphate. Particularly alanine and Glucose-6-phosphatase: glutamine, which are Converts glucose-6-phosphate released from muscle to free glucose, which is then protein breakdown. released into the bloodstream. Regulation: Gluconeogenesis is tightly Importance of regulated by hormonal and Gluconeogenesis metabolic signals: Maintaining Blood Insulin: Inhibits Glucose Levels: gluconeogenesis by Gluconeogenesis is decreasing the expression essential for maintaining of key gluconeogenic blood glucose levels enzymes. during fasting, Glucagon: Stimulates prolonged exercise, and gluconeogenesis by periods of low increasing the expression carbohydrate intake. of key gluconeogenic Energy Supply: Provides enzymes. a continuous supply of Cortisol: Enhances glucose for tissues that gluconeogenesis during rely heavily on glucose, stress by increasing the such as the brain and availability of substrates red blood cells. and the expression of gluconeogenic enzyme  Process of Glycolysis 1. Glucose Activation: 1. Step 1: Glucose is Glycolysis: The Breakdown phosphorylated by of Glucose Cleavage Phase: hexokinase to form Glycolysis is a fundamental Step 4: F1,6BP is split by glucose-6-phosphate metabolic pathway that aldolase into two three- (G6P), using one breaks down glucose to carbon molecules: molecule of ATP. produce energy. It occurs glyceraldehyde-3- 2. Step 2: G6P is in the cytoplasm of cells phosphate (G3P) and isomerized to fructose-6- and is the first step in both dihydroxyacetone phosphate (F6P) by aerobic and anaerobic phosphate (DHAP). phosphoglucose respiration. Here's a Step 5: DHAP is isomerase. detailed look at the converted to G3P by 3. Step 3: F6P is process and its triose phosphate phosphorylated by significance: isomerase, resulting in phosphofructokinase-1 two molecules of G3P. (PFK-1) to form fructose- 1,6-bisphosphate (F1,6BP), using another molecule of ATP. Energy Harvesting Phase: Step 6: G3P is oxidized by glyceraldehyde- 3-phosphate dehydrogenase to 1,3- Net Yield of Glycolysis bisphosphoglycerate (1,3BPG), producing ATP: 2 molecules of ATP NADH from NAD⁺. (4 produced, 2 Step 7: 1,3BPG is converted to 3- consumed) phosphoglycerate (3PG) by NADH: 2 molecules of phosphoglycerate kinase, generating one NADH molecule of ATP per G3P. Pyruvate: 2 molecules of Step 8: 3PG is converted to 2- pyruvate phosphoglycerate (2PG) by phosphoglycerate mutase. Step 9: 2PG is dehydrated by enolase to form phosphoenolpyruvate (PEP). Step 10: PEP is converted to pyruvate by pyruvate kinase, generating another molecule of ATP per G3P. Significance of Glycolysis 1. Energy Production: 1. Glycolysis provides a quick source of ATP, which is essential for cellular activities, especially in tissues with high energy demands like muscles and the brain. 2. Anaerobic Conditions: 1. In the absence of oxygen, glycolysis is the primary pathway for ATP production. Pyruvate is converted to lactate in anaerobic conditions, allowing glycolysis to continue. 3. Aerobic Respiration: 1. Under aerobic conditions, pyruvate produced in glycolysis is transported into the mitochondria for further oxidation in the citric acid cycle and oxidative phosphorylation, leading to the production of much more ATP. 4. Metabolic Intermediates: 1. Glycolysis provides intermediates for other metabolic pathways, such as the synthesis of amino acids, nucleotides, and lipids.  Hormonal Regulation 1. Insulin: 1. Produced by: Beta cells of the pancreas. 2. Function: Lowers blood glucose levels by promoting the uptake of glucose into cells, particularly muscle and fat cells, and by stimulating the conversion of glucose to glycogen in the liver The body (glycogenesis). regulates 2. Glucagon: blood glucose 1. Produced by: Alpha cells of the pancreas. levels through 2. Function: Raises blood glucose levels by stimulating the breakdown of glycogen to glucose in the a complex liver (glycogenolysis) and promoting the production of glucose from non-carbohydrate sources interplay of (gluconeogenesis). hormones and 3. Epinephrine (Adrenaline): physiological 1. Produced by: Adrenal glands. processes to 2. Function: Increases blood glucose levels by stimulating glycogenolysis and gluconeogenesis, maintain especially during stress or physical activity. homeostasis. 4. Cortisol: Here's how it 1. Produced by: Adrenal cortex. works: 2. Function: Increases blood glucose levels by promoting gluconeogenesis and reducing glucose uptake by cells. 5. Growth Hormone: 1. Produced by: Pituitary gland. 2. Function: Increases blood glucose levels by reducing glucose uptake by cells and promoting gluconeogenesis Physiological Processes 1. Glycogenesis: 1. Process: Conversion of glucose to glycogen for storage in the liver and muscles. 2. Stimulated by: Insulin. 2. Glycogenolysis: Feedback Mechanisms 1. Process: Breakdown of glycogen to Negative Feedback: The body uses negative feedback loops to maintain release glucose into the blood glucose levels within a narrow range. For example, high blood glucose bloodstream. levels stimulate insulin release, which lowers blood glucose, reducing the 2. Stimulated by: Glucagon and stimulus for further insulin release. epinephrine. Overall Balance 3. Gluconeogenesis: Homeostasis: The balance between insulin and counter-regulatory hormones 1. Process: Production of glucose (glucagon, epinephrine, cortisol, and growth hormone) ensures that blood from non-carbohydrate sources glucose levels remain stable, providing a continuous supply of energy to the such as amino acids and glycerol. body's cells 2. Stimulated by: Glucagon, cortisol, and growth hormone. 4. Glucose Uptake: 1. Process: Transport of glucose into cells for energy production or storage. 2. Stimulated by: Insulin.  Insulin Produced by: Beta cells of the pancreas. Primary Role: Lowers blood glucose levels. Roles of Glucagon Mechanism of Action: and Insulin in Glucose Uptake: Insulin facilitates the uptake of glucose into muscle and fat cells by promoting the Blood Glucose translocation of glucose transporter type 4 (GLUT4) to the cell membrane. Regulation Glycogenesis: Insulin stimulates the conversion of glucose to glycogen for storage in the liver and Insulin and muscles. glucagon are two Lipogenesis: Insulin promotes the conversion of excess glucose into fatty acids and their storage as key hormones triglycerides in adipose tissue. produced by the Protein Synthesis: Insulin facilitates the uptake of amino acids into cells, promoting protein pancreas that work synthesis. in tandem to Inhibition of Gluconeogenesis: Insulin inhibits the production of glucose from non-carbohydrate maintain blood sources in the liver glucose levels Glucagon within a narrow Produced by: Alpha cells of the pancreas. range. Here's how Primary Role: Raises blood glucose levels. they function Mechanism of Action: Glycogenolysis: Glucagon stimulates the breakdown of glycogen to release glucose into the bloodstream. Gluconeogenesis: Glucagon promotes the production of glucose from non-carbohydrate sources such as amino acids and glycerol in the liver. Lipolysis: Glucagon stimulates the breakdown of fats in adipose tissue, releasing free fatty acids into the bloodstream, which can be used for energy production. After a Meal (Postprandial State): 1. Increase in Blood Glucose: When you eat, especially foods rich in During Fasting (Postabsorptive State): carbohydrates, glucose is absorbed into 1. Decrease in Blood Glucose: When you fast, blood glucose levels the bloodstream from the digestive tract, gradually decrease as the body uses up the available glucose for causing a rise in blood glucose levels. energy. 2. Insulin Release: The pancreas detects 2. Glucagon Release: The pancreas releases glucagon in response the increase in blood glucose and to falling blood glucose levels. Glucagon stimulates the liver to releases insulin. Insulin facilitates the break down glycogen into glucose (glycogenolysis) and to uptake of glucose into cells, particularly produce glucose from non-carbohydrate sources muscle and fat cells, and promotes the (gluconeogenesis). storage of glucose as glycogen in the liver 3. Energy Mobilization: The body also begins to mobilize stored fats and muscles. for energy, converting triglycerides into free fatty acids and 3. Glucose Utilization: Cells use glucose for glycerol. Glycerol can be used in gluconeogenesis to produce energy production, and any excess glucose. glucose is stored as glycogen or 4. Maintenance of Blood Glucose: These processes help maintain converted to fat for long-term storage. blood glucose levels within a normal range, ensuring a 4. Return to Baseline: Blood glucose levels continuous supply of energy to vital organs, especially the brain. gradually return to baseline as glucose is taken up by cells and stored, typically within 2-3 hours after eating.. ❑ Self-Study Questions ❑ Clinical Significance ❑ What are the diagnostic criteria for diabetes mellitus? ❑ How do blood glucose levels vary among individuals with type 1 and 2 diabetes? ❑ Discuss the long-term complications associated with poorly controlled blood glucose levels. ❑ Determination of Blood Glucose ❑ What are the different methods for measuring blood glucose levels in the laboratory? ❑ Explain the principles behind common blood glucose measurement techniques, such as enzymatic assays and glucose oxidase methods. ❑ Discuss factors that can affect the accuracy of blood glucose measurements. ❑ Interpretation of Results ❑ What do fasting blood glucose levels indicate? ❑ How are oral glucose tolerance tests (OGTTs) used to diagnose diabetes mellitus? ❑ Interpret the significance of glycated hemoglobin (HbA1c) levels in diabetes management. ❑ Clinical Applications ❑ How do healthcare providers use blood glucose measurements to manage diabetes mellitus? ❑ Describe the importance of self-monitoring blood glucose (SMBG) in diabetes management. ❑ Discuss emerging technologies for continuous glucose monitoring (CGM) and their potential mpact on diabetes care.  1. Fasting Plasma Glucose. Random Plasma Glucose (FPG) Test Test Criteria: A fasting plasma Criteria: A random glucose level of 126 plasma glucose level of 4. Hemoglobin A1C mg/dL (7.0 mmol/L) or 200 mg/dL (11.1 (HbA1c) Test higher. mmol/L) or higher in a Criteria: An HbA1c level Procedure: Blood patient with classic The diagnostic of 6.5% (48 mmol/mol) glucose is measured symptoms of criteria for or higher. after an overnight fast hyperglycemia or diabetes Procedure: This test (no caloric intake for at hyperglycemic crisis. mellitus are measures the average least 8 hours). Procedure: Blood based on blood blood glucose levels over glucose is measured at glucose levels Oral Glucose Tolerance Test any time of the day, the past 2-3 months. and other (OGTT) regardless of when the indicators. Here Criteria: A 2-hour plasma person last ate. are the primary glucose level of 200 mg/dL criteria used to (11.1 mmol/L) or higher diagnose during a 75-g oral glucose diabetes: tolerance test. Procedure: Blood glucose is measured before and 2 hours after consuming a glucose-rich drink.  Type 2 Diabetes: Blood Glucose Levels in Nature: A metabolic disorder characterized by insulin resistance and, Type 1 vs. Type 2 Diabetes eventually, a decline in insulin production. The body either resists the effects of insulin or doesn't produce enough insulin to maintain Type 1 Diabetes: normal glucose levels. Nature: An autoimmune condition Blood Glucose Levels: Individuals with type 2 diabetes may have where the body's immune system elevated blood glucose levels, especially after meals. Over time, attacks insulin-producing beta cells in fasting blood glucose levels can also rise. The fluctuations are the pancreas, leading to little or no generally less severe than in type 1 diabetes but can still lead to insulin production. complications if not managed properly. Blood Glucose Levels: Individuals with Management: Often managed with lifestyle changes (diet and type 1 diabetes often experience exercise), oral medications, and sometimes insulin or other significant fluctuations in blood glucose injectable medications. Regular monitoring of blood glucose levels is levels. Without insulin, blood glucose also important levels can rise rapidly, leading to hyperglycemia. Insulin therapy is Comparison of Blood Glucose Targets: essential to manage these levels. Fasting Blood Glucose: Management: Requires regular Type 1 Diabetes: Typically 80-130 mg/dL (4.4-7.2 mmol/L). monitoring of blood glucose levels, Type 2 Diabetes: Generally 80-130 mg/dL (4.4-7.2 mmol/L). multiple daily insulin injections or an Postprandial (After Meals) Blood Glucose: insulin pump, and careful management Type 1 Diabetes: Less than 180 mg/dL (10.0 mmol/L) 1-2 hours after meals. of diet and physical activity. Type 2 Diabetes: Less than 180 mg/dL (10.0 mmol/L) 1-2 hours after meals. Poorly controlled blood glucose levels can lead to a range of serious long-term complications, affecting various organs and systems in the body. Here are some of the major complications: 1. Cardiovascular Disease Heart Disease: High blood glucose levels can damage blood vessels and the nerves that control the heart, increasing the risk of heart disease and heart attacks. Stroke: Poorly controlled diabetes can lead to atherosclerosis (hardening of the arteries), which increases the risk of stroke. 2. Neuropathy (Nerve Damage) Peripheral Neuropathy: Damage to the nerves in the extremities can cause pain, tingling, and loss of sensation, particularly in the feet and hands. Autonomic Neuropathy: This affects the nerves that control internal organs, leading to issues such as digestive problems, bladder dysfunction, and sexual dysfunction. 3. Nephropathy (Kidney Damage) Chronic Kidney Disease: High blood glucose levels can damage the kidneys' filtering system, leading to chronic kidney disease and, eventually, kidney failure. This may require dialysis or a kidney transplant. 4. Retinopathy (Eye Damage) Diabetic Retinopathy: Damage to the blood vessels in the retina can lead to vision problems and, in severe cases, blindness. Other Eye Conditions: Diabetes increases the risk of cataracts and glaucoma. Poorly controlled blood glucose levels can lead to a range of serious long-term complications, affecting various organs and systems in the body. Here are some of the major complications: 5. Foot Complications Ulcers and Infections: Poor blood flow and nerve damage can lead to foot ulcers and infections, which may require amputation if not properly managed. 6. Skin Conditions Infections: People with diabetes are more prone to bacterial and fungal skin infections. Other Skin Issues: Conditions such as diabetic dermopathy, necrobiosis lipoidica, and acanthosis nigricans can occur. 7. Dental Problems Gum Disease: High blood glucose levels can lead to gum disease and other oral health issues. 8. Mental Health Issues Depression and Anxiety: The stress of managing diabetes and its complications can contribute to mental health issues. 9. Increased Risk of Infections Weakened Immune System: Poorly controlled diabetes can weaken the immune system, making individuals more susceptible to infections. 10. Complications During Pregnancy Gestational Diabetes: Poorly controlled blood glucose levels during pregnancy can lead to complications for both the mother and the baby, including preeclampsia, premature birth, and birth defects There are several methods used to measure blood glucose levels in the laboratory, each with its own advantages and applications. Here are some of the most common methods: 1. Enzymatic Methods Glucose Oxidase Method: This method uses the enzyme glucose oxidase to catalyze the oxidation of glucose to gluconic acid and hydrogen peroxide. The hydrogen peroxide produced is then measured, which is proportional to the glucose concentration. Hexokinase Method: This method involves the phosphorylation of glucose by hexokinase to produce glucose-6- phosphate, which is then oxidized by glucose-6-phosphate dehydrogenase, producing NADH. The amount of NADH produced is proportional to the glucose concentration. 2. Chemical Methods O-Toluidine Method: This method involves the reaction of glucose with o-toluidine in an acidic medium to produce a green-colored complex, which is measured spectrophotometrically. This method is less commonly used due to its potential toxicity. 3. Electrochemical Methods Biosensors: These devices use glucose oxidase immobilized on an electrode. The enzyme catalyzes the oxidation of glucose, and the resulting current is measured, which is proportional to the glucose concentration. 4. Chromatographic Methods High-Performance Liquid Chromatography (HPLC): This method separates glucose from other components in the blood and measures it using a detector, such as a refractive index detector or a mass spectrometer. 5. Continuous Glucose Monitoring (CGM) CGM Devices: These devices use a small sensor inserted under the skin to measure glucose levels in the interstitial fluid continuously. The data is transmitted to a receiver or smartphone, providing real-time glucose monitoring Principles Behind Common Blood Glucose Measurement Techniques 1. Enzymatic Assays Enzymatic assays are widely used for measuring blood glucose levels due to their specificity and accuracy. These assays rely on enzymes that catalyze reactions involving glucose, producing measurable products. Glucose Oxidase Method: Principle: This method uses the enzyme glucose oxidase to catalyze the oxidation of glucose to gluconic acid and hydrogen peroxide. Reaction: Detection: The hydrogen peroxide produced is then measured, typically using a colorimetric or electrochemical method. In colorimetric assays, a chromogen reacts with hydrogen peroxide in the presence of peroxidase to produce a colored compound, which is measured spectrophotometrically. In electrochemical assays, the hydrogen peroxide is detected by an electrode, generating a current proportional to the glucose concentration. Principles Behind Common Blood Glucose Measurement Techniques Hexokinase Method: Principle: This method involves the phosphorylation of glucose by hexokinase to produce glucose-6-phosphate (G6P), which is then oxidized by glucose-6-phosphate dehydrogenase (G6PD) to produce NADH. Reaction: Detection: The amount of NADH produced is measured spectrophotometrically, as it absorbs light at a specific wavelength (340 nm), and its concentration is proportional to the glucose concentration. Principles Behind Common Blood Glucose Measurement Techniques 2. Electrochemical Methods Electrochemical methods are commonly used in portable glucose meters and continuous glucose monitoring systems. Biosensors: Principle: These devices use glucose oxidase immobilized on an electrode. The enzyme catalyzes the oxidation of glucose, producing hydrogen peroxide. Detection: The hydrogen peroxide is detected by an electrode, generating an electrical current proportional to the glucose concentration. This current is measured and converted into a glucose concentration reading Several factors can affect the accuracy of blood glucose measurements. Here are some key factors to consider: 1. Test Strip Issues Damaged or Expired Strips: Using damaged or outdated test strips can lead to inaccurate readings. Always check the expiration date and store strips properly. Compatibility: Ensure that the test strips are compatible with your specific glucose meter. 2. Environmental Conditions Temperature and Humidity: Extreme temperatures and high humidity can affect the accuracy of blood glucose meters and test strips. Keep your meter and strips at room temperature. Altitude: High altitudes can also impact readings, potentially leading to underestimation of blood glucose levels. 3. Skin Contaminants Substances on Skin: Alcohol, dirt, or other substances on your skin can affect the accuracy of the measurement. Wash and dry your hands thoroughly before testing. 4. Blood Sample Issues Insufficient Blood Sample: Not applying enough blood to the test strip can result in inaccurate readings. Ensure you use a generous drop of blood. Testing Site: Blood samples from alternate sites (e.g., forearm) may not be as accurate as fingertip samples, especially when blood glucose levels are changing rapidly. Several factors can affect the accuracy of blood glucose measurements. Here are some key factors to consider: 5. Hematocrit Levels Red Blood Cell Count: Variations in hematocrit levels (the proportion of red blood cells in your blood) can affect the accuracy of blood glucose measurements. Low hematocrit (anemia) or high hematocrit can lead to inaccurate results. 6. Interfering Substances Medications and Foods: Certain medications (e.g., paracetamol) and foods containing maltose or high levels of vitamin C can interfere with glucose measurements, leading to false results. 7. Meter Maintenance Monitor Problems: Ensure the test strip is fully inserted into the meter, and replace the batteries as needed. Regularly check and maintain your glucose meter to ensure it is functioning correctly. 8. Quality Control Control Solutions: Use control solutions to test your meter and strips periodically. This helps ensure that your meter is providing accurate reading Fasting Blood Glucose Levels: What They Indicate Fasting blood glucose levels are measured after an individual has not eaten for at least 8 hours. This test is commonly used to assess how well the body manages blood sugar levels and can indicate various health conditions: Normal Range Normal Fasting Blood Glucose: 70-99 mg/dL (3.9-5.5 mmol/L) Indication: Indicates normal glucose metabolism and effective insulin function. Prediabetes Prediabetes Range: 100-125 mg/dL (5.6-6.9 mmol/L) Indication: Suggests impaired fasting glucose (IFG), a condition where blood glucose levels are higher than normal but not high enough to be classified as diabetes. It indicates an increased risk of developing type 2 diabetes and cardiovascular disease. Diabetes Diabetes Range: 126 mg/dL (7.0 mmol/L) or higher on two separate tests Indication: Indicates diabetes mellitus, a condition where the body either does not produce enough insulin (type 1 diabetes) or cannot effectively use the insulin it produces (type 2 diabetes). This requires medical management to prevent complications. Fasting Blood Glucose Levels: What They Indicate Hypoglycemia Low Fasting Blood Glucose: Below 70 mg/dL (3.9 mmol/L) Indication: Indicates hypoglycemia, a condition where blood glucose levels are too low. This can be caused by excessive insulin, certain medications, prolonged fasting, or other medical conditions. Symptoms may include shakiness, sweating, confusion, and in severe cases, loss of consciousness. Significance Diagnosis: Fasting blood glucose levels are crucial for diagnosing diabetes and prediabetes. Monitoring: Regular monitoring helps manage diabetes and prevent complications by ensuring blood glucose levels remain within the target range. Health Assessment: Provides insight into overall metabolic health and the risk of developing diabetes-related complications Oral Glucose Tolerance Test (OGTT) for Diagnosing nterpretation of Results​ Diabetes Mellitus Normal:​ The Oral Glucose Tolerance Test (OGTT) is a Fasting blood glucose: Less than 100 mg/dL (5.6 mmol/L)​ diagnostic tool used to assess how well the body 2-hour blood glucose: Less than 140 mg/dL (7.8 mmol/L)​ processes glucose. It is particularly useful for Prediabetes:​ diagnosing diabetes mellitus and other conditions Fasting blood glucose: 100-125 mg/dL (5.6-6.9 mmol/L)​ related to glucose metabolism. Here's how it 2-hour blood glucose: 140-199 mg/dL (7.8-11.0 mmol/L)​ works: Diabetes:​ Procedure Fasting blood glucose: 126 mg/dL (7.0 mmol/L) or higher​ 1. Preparation: The patient is required to fast for at 2-hour blood glucose: 200 mg/dL (11.1 mmol/L) or higher​ least 8-12 hours before the test. This means no food or drink, except water, during this period. 2. Baseline Measurement: A blood sample is taken Significance​ to measure the fasting blood glucose level. Diagnosis: The OGTT helps diagnose diabetes by revealing how 3. Glucose Drink: The patient consumes a glucose- the body handles glucose over time. Elevated blood glucose rich drink, typically containing 75 grams of levels at the 2-hour mark indicate impaired glucose tolerance glucose. or diabetes.​ 4. Blood Samples: Blood samples are taken at Gestational Diabetes: The OGTT is also used to diagnose regular intervals, usually at 30 minutes, 1 hour, gestational diabetes in pregnant women, typically performed and 2 hours after consuming the glucose drink. between 24 and 28 weeks of pregnancy​ These samples measure how the blood glucose levels change over time. I Significance of Glycated Hemoglobin (HbA1c) Levels in Diabetes Management Glycated hemoglobin (HbA1c) is a crucial marker used to assess long-term blood glucose control in individuals with diabetes. Here's why it is significant: 1. Long-Term Glucose Control Average Blood Glucose: HbA1c reflects the average blood glucose levels over the past 2-3 months. This is because glucose molecules attach to hemoglobin in red blood cells, and these cells have a lifespan of about 120 days. Stability: Unlike daily blood glucose measurements, which can fluctuate due to various factors, HbA1c provides a more stable and comprehensive picture of glucose control. 2. Diagnostic Tool Diagnosis: HbA1c is used to diagnose diabetes and prediabetes. An HbA1c level of 6.5% (48 mmol/mol) or higher indicates diabetes, while a level between 5.7% and 6.4% (39-47 mmol/mol) indicates prediabetes. Screening: It is also used for screening individuals at risk of developing diabetes. Significance of Glycated Hemoglobin (HbA1c) Levels in Diabetes Management Glycated hemoglobin (HbA1c) is a crucial marker used to assess long-term blood glucose control in individuals with diabetes. Here's why it is significant: 3. Monitoring and Management Treatment Efficacy: Regular HbA1c testing helps healthcare providers assess the effectiveness of diabetes treatment plans, including medication, diet, and lifestyle changes. Adjustments: Based on HbA1c levels, treatment plans can be adjusted to improve blood glucose control and prevent complications. 4. Risk Assessment Complications: Higher HbA1c levels are associated with an increased risk of diabetes-related complications, such as cardiovascular disease, neuropathy, nephropathy, and retinopathy. Keeping HbA1c levels within the target range reduces the risk of these complications. 5. Target Levels Individualized Goals: The target HbA1c level may vary depending on individual factors such as age, duration of diabetes, and presence of other health conditions. Generally, a target HbA1c level of less than 7% (53 mmol/mol) is recommended for most adults with diabetes, but this can be adjusted based on individual needs. Healthcare providers use blood glucose measurements to manage diabetes mellitus by monitoring and adjusting treatment plans to maintain optimal blood glucose levels. Here are some key ways they use these measurements: 1. Diagnosis Initial Diagnosis: Blood glucose measurements, including fasting plasma glucose (FPG), oral glucose tolerance test (OGTT), and HbA1c levels, are used to diagnose diabetes and prediabetes. 2. Monitoring Daily Monitoring: Patients with diabetes are often advised to monitor their blood glucose levels daily using a glucometer. This helps track how well their blood glucose is controlled and identify patterns. Continuous Glucose Monitoring (CGM): CGM devices provide real-time glucose readings throughout the day and night, offering a comprehensive view of glucose trends and helping to prevent hypo- and hyperglycemia. 3. Treatment Adjustments Medication Management: Blood glucose measurements help healthcare providers adjust medications, including insulin and oral hypoglycemic agents, to achieve target glucose levels. Insulin Dosing: For patients on insulin therapy, blood glucose readings guide the dosing and timing of insulin injections to prevent fluctuations in blood glucose levels. Lifestyle Modifications Diet and Exercise: Blood glucose measurements inform dietary and physical activity recommendations. Patients can see the impact of different foods and exercise on their blood glucose levels, helping them make informed choices. Education: Healthcare providers use blood glucose data to educate patients about managing their condition, including recognizing the signs of hypo- and hyperglycemia and how to respond. 5. Assessing Long-Term Control HbA1c Levels: Regular HbA1c tests provide an overview of average blood glucose levels over the past 2-3 months, helping to assess long-term glucose control and the risk of complications. Adjusting Goals: Based on HbA1c and daily glucose measurements, healthcare providers can set and adjust individualized treatment goals. 6. Preventing Complications Early Detection: Regular monitoring helps detect early signs of complications, such as neuropathy, retinopathy, and nephropathy, allowing for timely intervention. Risk Management: Blood glucose measurements help manage the risk of acute complications like diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS). ealthcare providers use blood glucose measurements to manage diabetes mellitus by monitoring and adjusting treatment plans to maintain optimal blood glucose levels. Here are some key ways they use these measurements: 1. Diagnosis Initial Diagnosis: Blood glucose measurements, including fasting plasma glucose (FPG), oral glucose tolerance test (OGTT), and HbA1c levels, are used to diagnose diabetes and prediabetes. 2. Monitoring Daily Monitoring: Patients with diabetes are often advised to monitor their blood glucose levels daily using a glucometer. This helps track how well their blood glucose is controlled and identify patterns. Continuous Glucose Monitoring (CGM): CGM devices provide real-time glucose readings throughout the day and night, offering a comprehensive view of glucose trends and helping to prevent hypo- and hyperglycemia. 3. Treatment Adjustments Medication Management: Blood glucose measurements help healthcare providers adjust medications, including insulin and oral hypoglycemic agents, to achieve target glucose levels. Insulin Dosing: For patients on insulin therapy, blood glucose readings guide the dosing and timing of insulin injections to prevent fluctuations in blood glucose levels. 4. Lifestyle Modifications Diet and Exercise: Blood glucose measurements inform dietary and physical activity recommendations. Patients can see the impact of different foods and exercise on their blood glucose levels, helping them make informed choices. Education: Healthcare providers use blood glucose data to educate patients about managing their condition, including recognizing the signs of hypo- and hyperglycemia and how to respond. 5. Assessing Long-Term Control HbA1c Levels: Regular HbA1c tests provide an overview of average blood glucose levels over the past 2-3 months, helping to assess long-term glucose control and the risk of complications. Adjusting Goals: Based on HbA1c and daily glucose measurements, healthcare providers can set and adjust LABORATORY ACTIVITY 19A QUALITATIVE RECTION of TOCOPHEROL 1. Introduction Vitamins Clinical Significance ❑ Vitamins are essential organic compounds that are ❑ Vitamin deficiencies can lead to various health required in small quantities for various biochemical problems and diseases. processes in the body. ❑ For example, vitamin D deficiency can result in ❑ They play crucial roles in metabolism, growth, bone disorders like rickets or osteoporosis, development, and maintaining overall health. while vitamin C deficiency can cause scurvy. ❑ There are two main types of vitamins: fat-soluble ❑ Conversely, excessive intake of certain vitamins can vitamins (A, D, E, and K) and water-soluble vitamins lead to toxicity and adverse effects. (B-co