CLICHM1 MIDTERMS – P2 INSTRUMENTATION, CHO, DM | BY: PEM GANDA <3

Part II Instrumentation Application: Metabolic Pathway

Electrochemistry • Allows definitive identification when used on samples eluting GC/HPLC Galactose, Fructose => Glucose
- Measurement of current/voltage by ions • MS + GC = Drug testing analysis (gold standard) Glucose + ATP =Hexokinase/Glucokinase=> Glucose-6-phosphate + ADP
Techniques: Glucose-6-phosphate enters 3 pathways:
CARBOHYDRATES (CHO)
Potentiometry 1. Glycolysis/Embden-Meyerhof PW
Function:
• Measure potential (voltage) between 2 electrodes •
• Major energy source (Glucose) Generate ATP; Products are Lactate and Pyruvate
• pH, pCO2, Na, Ca, K, NH
• Storage form of energy (Glycogen – stored in liver and muscles 2. Hexose Monophosphate PW
Amperometry •
• Components of cell membranes (Glycoproteins) Generate NADPH; Produces reduced Glutathione (anti-oxidant)
• Measure current flow by oxidation-reduction reaction
• Structural components of plants (cellulose) bacteria, & insects (chitin) 3. Glycogenesis
• pO2 (Clark electrode), glucose, peroxidase • Glucose => Glycogen
— Can be reducing (with free ketone/aldehyde (-OH)/hydroxyl group) or
➢ arterial blood gas (ABG): Arterial Blood | Anti-coagulant:
non-reducing (no free hydroxyl group) ➢ Glycolysis – metabolism of glucose molecule to pyruvate/lactate for
Heparin
Coulometry Types according to the number of units production of energy (ATP)
• electrochemical titration (titrant) Monosaccharide (Glucose, Fructose, Galactose) ➢ Glycogenolysis – breakdown of glycogen to glucose use as energy
• Cl (chloride) Disaccharide- 2 mono joined by Glycosidic bond ➢ Glycogenesis – conversion of glucose to glycogen for storage
Voltammetry • Lactose – glucose + galactose ➢ Gluconeogenesis – form glucose-6-phosphate from non-CHO sources
• Maltose – glucose + glucose ➢ Lipogenesis- convert carbohydrates to fatty acids
• Potential applied to electrochemical cell & resulting current measured
• Sucrose – glucose + fructose ➢ Lipolysis – decomposition of fats
• Anodic strip voltammetry (lead & iron)
Oligosaccharide – 3-10 units Regulation of Carbohydrate Metabolism
Electrophoresis Polysaccharide – more than 10 (Cellulose, starch, glycogen) 1. Insulin (Type I & II DM)
• Separate charged compounds based on electrical charge
Carbohydrates Metabolism • Produced by beta cells of islet of Langerhans in pancreas
➢ (+) => cathode (negative)
Mouth Saliva (Amylase/Ptyalin) • Prepoinsulin => Proinsulin => Insulin
➢ (-) => anode (positive)
• ➢ C-peptide: a marker of insulin production
• For DNA, RNA, & Protein analysis (all are negatively charged; anode) Mechanical digestion: chewing, swallowing
• Chemical digestion: carbohydrates, fats • Target: cells of the body
Components:
• Only hormone that has a hypoglycemic effect (lowers sugar level)
• Driving force – electrical power (cathode & anode) Stomach (no CHO metabolism)
• Physiologic effect of Insulin (Lower blood sugar)
• Support medium Mechanical digestion: peristaltic mixing, propulsion
• Chemical digestion: proteins, fats 1. Increase utilization of glucose of cells by increasing cellular uptake &
➢ Filter paper
• hepatic glycolysis
➢ Agarose (most commonly used; derived from red algae) Absorption: lipid-soluble substance (alcohol, aspirin)
2. Increase glycogenesis and inhibit glycogenolysis
➢ Cellulose acetate Small intestine (Pancreatic amylase/amylopsin)
3. Inhibits gluconeogenesis
➢ Polyacrylamide • Mechanical digestion: mixing, propulsion (segmentation)
4. Stimulate lipogenesis while inhibiting lipolysis
• Buffer • Chemical digestion: carbohydrates, fats, polypeptides, nucleic acids
5. Stimulate protein synthesis & stimulate uptake of amino acids into
➢ Tris-acetic EDTA (DNA analysis) • Absorption: peptides, amino acids, glucose, fructose, fats, water, minerals, muscles
➢ Tris-borate EDTA (DNA & RNA) vitamins
1. Provides electric power (force molecules to move in poles) Large intestine (no chemical digestion except bacteria) 2. Glucagon (Hyperglycemic effect – increase sugar level)
2. Maintain pH of solution (isotonic; not acidic/base) • Mechanical digestion: segmental mixing, propulsion • Alpha cells of islet of Langerhans (pancreas)
• Sample – molecule separated according to size and charge • Absorption: ions, water, minerals, vitamins, organic molecules • Target: liver
• Detecting system – densitometry • Glucose =>Glycogen = Increase Glucose level
1. Carbohydrates in diet constitute about 50% of calories:
▪ Dye used: ethidium bromide Physiologic effect of Glucagon
a. Starch – 60%
▪ Shorter size (small) = fast to travel 1. Promotes liver glycogenolysis
b. Sucrose – 30%
▪ Larger size = slow to travel 2. Increase gluconeogenesis
c. Lactose – 5%
3. Inhibits glycolysis
Chromatography d. Other sugar – 5%
• Separate complex mixtures on basis of different physical attraction e. Cellulose – part of dietary fiber Other hyperglycemic hormones
between individual compounds and stationary phase of system 2. Salivary amylase (ptyalin) and pancreatic amylase (amylopsin) break A. Cortisol
Components: down polymer polysaccharides to disaccharide B. Somatostatin
• Mobile phase – carry complex mixture (Gas/Liquid) 3. Disaccharides are further hydrolyzed to monosaccharides by enzyme C. Growth hormone
• Stationary phase – mobile phase flows (solid/liquid) disaccharidases D. Thyroid hormone
• Column – holds the stationary phase Disaccharide Enzyme (Disaccharidases) Product (Monosaccharide) E. Catecholamine
• Eluate – separated components Sucrose Sucrase Glucose + fructose ➢ NADPH – Nicotinamide adenine dinucleotide phosphate
Chromatographic Procedures Lactose Lactase Glucose + galactose ➢ Sugar for brain – glucose
I. Thin-layer Chromatography Maltose Maltase Glucose + glucose ➢ Also known as table sugar – sucrose
II. High-Performance Liquid Chromatography (HPLC) – use pressure 4. Monosaccharides are absorbed by the gut via Active transport (glucose ➢ Sweetest sugar and use for flavoring – fructose
for fast separation + galactose) or facilitated diffusion (fructose). They are then transported
HYPERGLYCEMIA
III. Gas chromatography (GC) – separate mixture of volatile compounds into the liver (portal circulation)
5. Glucose is the only CHO used directly for energy • increased plasma glucose levels
Mass Spectrometry (MS) 6. After glucose enters a cell, it undergoes phosphorylation to glucose-6- • caused by an imbalance of hormones
• Sample in a MS is 1st volatilized & ionized to form charged molecular ions phosphate through hexokinase and glucokinase • normally: insulin is secreted: enhances entry of glucose into the liver,
and fragments that are separated according to mass-to-charge (m/z) ratio muscle, and adipose, and alters glucose metabolic pathways

1|P a g e
 CLICHM1 MIDTERMS – P2 INSTRUMENTATION, CHO, DM | BY: PEM GANDA <3
Laboratory Findings in Hyperglycemia Type 1 Diabetes Mellitus • FPG ≥ 126 mg/dL (7.0 mmol/L). Fasting is defined as no caloric intake for at least
1. increased glucose in plasma and urine 8 hours
2. increased urine specific gravity • 2-H PG ≥ 200 mg/dL (11.1 mmol/L) during an OGTT. The test should be
performed by WHO, using a glucose load containing the equivalent of 75g of
3. increased serum and urine osmolality
anhydrous glucose dissolved in water.
4. ketonemia and ketonuria • A1C ≥ 6.5% (48 mmol/mol). The test should be performed using a method that is
5. decreased blood and urine pH (acidosis) NGSP-certified and standardized to the DCCT assay
6. electrolyte imbalance • In a patient with classic symptoms of hyperglycemia/hyperglycemic crisis,
Diabetes Mellitus random plasma glucose ≥200 mg/dL (11.1 mmol/L)
• Risk for diabetic ketoacidosis
o Heterogeneous group of multifactorial, polygenic syndromes characterized OGTT (Oral Glucose Tolerance Test)
• Ketone bodies
by elevated fasting blood glucose caused by absolute deficiency in insulin ➢ Beta-hydroxybutyric acid • FBG after an overnight fast
Categories (ADA/WHO) ➢ Acetoacetic acid • Glucose levels 1 hour & 2 hours after glucose load measured
1. Type 1 Diabetes Mellitus – absolute deficiency of insulin caused by ➢ Acetone (least abundant) • Use: detection of GDM
autoimmune diseases on beta cells of the pancreas Other specific types of diabetes PATIENT PREPARATION:
2. Type 2 Diabetes Mellitus – a combination of insulin resistance and 1. 3 days of unrestricted diet
• Associated with certain conditions (secondary): 2. Avoid medications that will interfere with the test
dysfunctional beta cells ➢ Genetic defects of beta-cell function/insulin action 3. No beverages
3. Other specific types of diabetes ➢ Pancreatic disease 4. No alcohol / no cigarettes
• monogenic diabetes syndromes (e.g., neonatal diabetes & ➢ Diseases of endocrine origin, drug-, or chemical-induced insulin 5. Overnight (8 – 14 hour) fast
maturity-onset diabetes of the young [MODY] receptor abnormalities A. 2-hour Postprandial Test
• diseases of the exocrine pancreas (e.g., cystic fibrosis) ➢ Certain genetic syndromes • Dose: a gram/kg body weight.
• drug- or chemical-induced diabetes (e.g., with glucocorticoid use) • Normal: peak value in 30 mins. (~150 mgs%); back to normal in 2 hrs.
4. Gestational Diabetes Mellitus – seen in pregnant women during Gestational Diabetes Mellitus (GDM) B. 5-hour Postprandial Test
pregnancy. Diabetes diagnosed in the 2nd or 3rd trimester of pregnancy • Glucose intolerance with onset or 1st recognition during pregnancy • Dose: 75 grams (WHO 1985)
that was not overt diabetes before gestation • Due to metabolic and hormonal changes • Normal: back to normal in 5 hours
ADA – American Diabetes Association • A large % of patients develop DM within 5-10 years ▪ Done if the value of 2-hr PPT is >140 – 160 mgs%
o Set guidelines for the early detection of DM • Mothers with DM increased risk for RDS, hypocalcemia, hyperbilirubinemia
• Screening: 2-hour OGTT using a 75g glucose load Intravenous Glucose Tolerance Test (IVGTT)
Type 1 Diabetes Type 2 Diabetes Indications:
➢ Fasting 8-14 hours
Other Names Juvenile Onset DM Adult-Onset DM ➢ 2-hour OFTT: Initial, 1st hour, 2nd hour • Poor absorption of ingested CHO
Insulin Dependent DM Non-insulin Dependent Standard: 70-100 (Glucose) • History of GIT surgery
• Kapag damaged DNA • Brain: most common consumer of glucose • Dose: 0.5g/kg (25 g/dL solution given intravenously)
pancreas – cannot • Erythrocyte: 2nd consumer of glucose Insulin tolerance test
produce insulin • Retinal cells (in eyes) • Dose: 0.1 unit/kg
Frequency 5-10% 90-95% 2 types of glucose load • Normal: 30 mins, 50% decrease in FBS level; back normal on 2nd hr.
Age of onset Common in children & young Common with advancing - 75g and 100g (amount of glucose present) CLINICAL SIGNIFICANCE:
adults age 1. Insulin Resistance
HYPOGLYCEMIA
Risk Factors Genetic, autoimmune, Genetic, obesity. • Delayed decrease in BGL
- results from an imbalance between glucose utilization and production
environmental (viral infection) Sedentary lifestyle, • DM, hyperadrenalism, Acromegaly
- observable symptoms appear at about 50-55 mg/dl
HLA DR3/4 race/ethnicity 2. Hypoglycemic Unresponsiveness
Symptoms:
Autoantibodies • Normal fall of BGL but delay in regaining normal value
A. Neurogenic
- Anti-islet • Addison’s disease, hypofunction of anterior PG, hyperinsulinism, Von
- triggered by ANS; tremulousness, palpitations, anxiety, diaphoresis,
cytoplasmic Gierke’s disease
hunger, paresthesia
antibody
B. Neuroglycopenic Tolbutamide Tolerance Test
- Insulin
- CNS hypoglycemia; dizziness, tingling, difficulty concentrating, blurred • Orinase or Tolbutamide
autoantibodies
vision - confusion, behavioral changes, seizure, coma ▪ Stimulates pancreas to produce insulin
- Anti-GAD (glutamic *Historically: post-absorptive (fasting) and postprandial (reactive) hypoglycemia
acid decarboxylase) ▪ Evaluate hypoglycemia caused by insulinoma
Pathogenesis Destruction of pancreatic beta No autoimmunity Diagnostic Criteria for Diabetes Mellitus (for 75g of glucose load) • Normal: 30 mins, 50% decrease in FBS level; back normal on 2nd hour
Fasting plasma 2-hour plasma glucose HbA1c
cells, usually autoimmune Insulin resistance & Epinephrine tolerance test
glucose level (after 75g load)
progressive insulin • Serves as an index of the quantity & availability of glycogen
deficiency mg/dL mmol/L mg/dL mmol/L %
Normal <100 <5.6 <140 <7.8 • Interpretation: Peak value after 30 mins.; normal within 2 hours
C-peptide Very low or undetectable Detectable
levels Pre-diabetes 5.7-6.4 • 35 – 45 mgs% increase in conc. between 45 – 60 mins.
Plasma Low to absent High in early disease; Impaired fasting 100-125 5.6-6.9 Lactose tolerance test
insulin low to absent in disease glucose • Detects the presence of mucosal lactase
Impaired glucose 149-199 7.8-11.0
of long duration • Causes of Lactose Intolerance:
tolerance
Ketoacidosis Prone to ketoacidosis and Not prone to
Diabetes mellitus
▪ Deficiency of lactase
>126 >7.0 >200 >11.1 >6.5
diabetic complications ketoacidosis ▪ Inhibition to lactase activity (e.g., intestinal disorders)

2|P a g e
 CLICHM1 MIDTERMS – P2 INSTRUMENTATION, CHO, DM | BY: PEM GANDA <3
Diagnosis of Gestational Diabetes Mellitus • type 1 DM: acute illness, stress, pregnancy, hyperglycemia of >300mg/dL or 1. Oxidation-Reduction Method (Redox)
100g OGTT plasma glucose 75g OGTT plasma glucose with signs of ketoacidosis ▪ LEO – Loss of electron (Oxidation)
mg/dL mmol/L mg/dL mmol/L ▪ DKA: serious and potentially fatal hyperglycemic condition ▪ GER – Gain of electron (Reduction)
Fasting >95 >5.3 >95 >5.3 ✓ nausea, vomiting, abdominal pain, electrolyte disturbances & A. Alkaline Copper Reduction Method
1 hour >180 >10.0 >180 >10.0 dehydration
I. Folin-Wu: phosphomolybdic acid => phosphomolybdenum blue
2 hours >155 >8.6 >155 >8.6 • specimen considerations:
• total reducing substances (true reducing substances and
3 hours >140 >7.8 ▪ Fresh serum or urine
saccharoid) measured
GDM is diagnosed if >2 plasma glucose levels are exceeded ▪ Tightly stoppered and assayed ASAP
II. Nelson-Somogyi: arsenomolybdic acid => arsenomolybdenum blue
• lab methods:
Glycosylated Hemoglobin (HbA1c) • barium sulfate enables measurement of true reducing
• Rate of formation of HbA1c is proportional to average blood glucose a. Gerhardt's: acetoacetic acid + ferric chloride ---> red color
substances only
concentration over 3 months b. acetoacetic acid + sodium nitroprusside ---> purple color
III. Neocuproine method — Campbell and King method
➢ 3-6 months; provides an index of average BGL over the past Enzymatic method: acetoacetic acid + NADH + H =β-HBDH=> NAD + β-hydroxybutyric ac • neocuproine (7,9- dimethyl-1,10-phenanthroline) =>
2-4 months Fructosamine (Glycated Albumin) cuprous-neocuproine complex (yellow-orange)
• Every 1% increase in HbA1c is 35mg/dL change in plasma glucose • Monitoring glucose control over the past 2-3 weeks IV. Benedict’s method
• ADA recommends that HbA1c be tested at least twice a year to • Albumin has a half-lifespan 20 days in circulation
monitor long-term glycemic control B. Alkaline Ferric Reduction Method (Hagedorn-Jensen)
• Affected by albumin levels, false decrease in px with hypoalbuminemia • reverse colorimetric method
• Specimen: EDTA - Whole blood => hemolysate
➢ Values for HbA1 & HbA1c have a high degree of correlation Hypoglycemia • ferricyanide --- > ferrocyanide
• False decrease: decreased RBC lifespan • 50-55mg/dL, glucagon, and other glycemic • more specific than copper reduction
factors are released. Observable symptoms 1. Johnson method
➢ 120 days lifespan of RBC
appear 2. Folin/Prussian blue method
• Glucose + amino group of hemoglobin = ketoamine (rate of formation 3. Hagedorn-Jensen method
is directly proportional to plasma glucose concentration) • Causes secondary to other illnesses & resolve
METHODS for MEASUREMENT of Hemoglobin A1C: themselves when primary disorder is treated 2. Condensation Method (Dubowski)
1. ION-EXCHANGE CHROMATOGRAPHY • Examples: A. Glucose Oxidase
• Cation-exchange resin or carboxymethyl cellulose resin ➢ Insulinoma
2. HPLC ➢ Various liver disorders
• Reference method ➢ Gastrointestinal disorders & surgery
➢
An enzymatic method. Most specific enzymes reacting with B-D-
3. COLORIMETRY Warning signs and symptoms are all related to the central nervous system:
glucose
• Hb A1c --- acid ----5-HMF ▪ Increased hunger
▪ Sweating ➢ Reaction can be monitored by:
4. RADIOIMMUNOASSAY (RIA)
▪ Nausea and vomiting 1. Polarographically - measure rate of disappearance of oxygen
• Antibodies against Hb A1c (sheep antiserum) ▪ Dizziness using an oxygen electrode
5. ELECTROPHORESIS ▪ Nervousness and shaking ▪ Self-Monitoring of Blood Glucose: point of care device
• Citrate agar electrophoresis (pH 6.0-6.2) ▪ Blurring of speech and sight - recommended for type 1 diabetics
• Good resolution of Hb A & Hb A1 ▪ Mental confusion
- to maintain levels as close to normal as possible
6. ISOELECTRIC FOCUSING Specimen considerations and patient preparation - whole blood glucose testing using capillary blood
• Scanned on high-resolution microdensitometer • Possible specimens = whole blood, plasma, urine, CSF, serous as sample
• Hb A1c is adequately resolved from HbA1a, A1b, S and F fluid, synovial fluid 2. Colorimetrically – side reaction that consumes H2O2
7. AFFINITY CHROMATOGRAPHY • Standard clinical specimen = Fasting venous plasma ➢ Mutarotase: added to convert alpha to beta glucose
• Use of affinity gel columns • Fasting blood sugar should be obtained after 8-10 hours of fasting Methods to quantify H2O2:
Microalbuminuria ➢ Lipids: 10-12 hours 1. Gochman and Schmitz: 3-methyl-2-benzathiazolinone hydrazone + N, N-
• useful to assist in diagnosis at an early stage and before the • Whole blood glucose levels = 10-15% lower than plasma levels dimethylaniline => indamine dye (590-66- nm)
development of proteinuria (> 0.5g/day) • Glucose is metabolized at room temp at 7mg/dL/hr 2. Trinder: p-amino phenazone + phenol => purple (quinonimine)
C peptide • 4C, glucose decreases by approximately 2mg/dL/hr 3. Miskieweis: 2,2-azine-di-3-ethyl bezothiazoline-6-sulfonic acid => colored
• reflects pancreatic secretion of insulin • Evacuated tube = gray top (glucose determination) chromogen
• proinsulin cleaved to give C peptide and insulin ➢ Potassium oxalate (anti-coagulant) B. Hexokinase
• reflects endogenous insulin production if a patient is taking insulin ➢ Sodium fluoride (inhibits breakdown of glucose;
glycogenolysis)
DETECTION OF ACUTE CRISIS SITUATIONS
Ketone measurement • 10% contamination with 5% dextrose (D5W) will elevate the glucose
• ketone bodies: produced by the liver through the metabolism of fatty
in a sample by 500mg/dL or more ➢ More accurate, less interferences than glucose oxidase methods
acids provide a ready energy source from stored lipids at times of low Methods of Glucose Measurements because the coupling reaction using GGPDH is highly specific;
CHO availability • Chemical Methods & Enzymatic Methods therefore, less interference than coupled glucose oxidase procedures
▪ NADPH –Strong absorbance at 340nm
• 3 types: Principle of Chemical Methods ➢ REFERENCE method - not affected by uric acid and ascorbic acid
▪ acetone (2%) ➢ Glucose and other CHO can convert cupric in alkaline sol’n to cuprous ➢ False decrease: hemolyzed sample and elevated bilirubin levels
▪ acetoacetic acid (20%) ➢ Glucose & other CHO are reducing sugars (except sucrose)
➢ Can also be used for urine, CSF and serous fluids
▪ 3-beta-hydroxybutyric acid (78%) ▪ Reduction: gain of electron
• ketonemia and ketonuria ▪ Reduced substance: receives electrons
▪ Reducing substance: gives electrons

3|P a g e