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CARBOHYDRATES general formula: Cx (H2O)y Functions:

THREE CLASSES:  Major energy sources

 Essential structural
a.) MONOSACCHARIDES – 1 saccharide (“simple sugar”) unit components
Examples:
Glucose/Dextrose – dextrorotatory – rotate polarized light to the right
Reducing sugars with
Fructose/Levulose – levorotatory – rotate polarized light to the left
reducing properties
Galactose
b.) DISACCHARIDES – 2 saccharide units
Examples:
Maltose = glucose + glucose ~maltase Disaccharases – enzymes that hydrolyze disaccharides;
Lactose = glucose + galactose ~lactase breaks or splits them into two monosaccharides and
Sucrose = glucose + fructose ~sucrase producing a water molecule ion the process
c.) POLYSACCHARIDES - 3 or more saccharide units
Examples:
Starch – storage form of glucose in plants
– Main carbohydrate in diet
– digested by AMS (amylase) in saliva and pancreas
Glycogen – storage form of glucose in animals; stored in liver
– “Quick energy” – easily converted to glucose
Cellulose – major structural components in plants; i.e. fibers in fruits and vegetables
– No nutritional values and not degraded by enzymes
– Responsible for normal functioning of the human intestine

HORMONAL CONTROL OF CARBOHYDRATE METABOLISM

1. GLYCOLYSIS (in the muscles) – breakdown of glucose to lactate and pyruvate forming CO2, H2O and energy
2. GLYCOGENESIS (in the liver) – anabolic formation/synthesis of glycogen from glucose; due to high glucose levels
3. GLYCOGENOLYSIS (in the liver) – breakdown of glycogen to glucose
4. GLUCONEOGENESIS – formation of glucose from non-carbohydrate sources (e.g., fatty acids, amino acids, etc.)

*HORMONES INFLUENCING CARBOHYDRATE METABOLISM

1. INSULIN – from the β cells of the pancreas; lowers glucose levels; promotes glycolysis and glycogenesis
2. GLUCAGON – from the α cells of the pancreas; increases glucose levels; promotes glycogenolysis
3. CORTISOL – from adrenal cortex; increases glucose levels; promotes gluconeogenesis
4. SOMATOSTATIN – from the δ cells of the pancreas; regulates balance of insulin and glucagon

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RELATED DISEASES

INSULINOMA – characterized by hypoglycemia; increase of insulin secretion due to overproduction of β cells in the pancreas;
causes a pancreatic tumor and lead to cancer

DIABETES MELLITUS – characterized by hyperglycemia; most common disease associated with Carbohydrate Metabolism; may be
of Type 1, Type 2 or Gestational

DIABETES MELLITUS (DM)

- The most common disease associated with Carbohydrate Metabolism
- A metabolic disorder characterized by hyperglycemia due to defect in insulin, action, secretion, or both

TYPE 1 DIABETES MELLITUS

 IDDM – Insulin Dependent Diabetes Mellitus

 Cellular-mediated autoimmune destruction of the β cells of the pancreas, causing an absolute deficiency of insulin
secretion
 Constitutes only 10-20% of all cases of diabetes and has juvenile onset i.e., commonly occurring in childhood and
adolescence
 Increased danger of ketosis – accumulation of ketone bodies
 Treatment is through regular insulin administration

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TYPE 2 DIABETES MELLITUS

 NIDDM – Non-Insulin Dependent Diabetes Mellitus

 Characterized by hyperglycemia as a result of an individual’s resistance to insulin
 Constitutes the majority of diabetes cases
 Most patients in this type are obese or have an increased percentage of body fat distribution in the abdomen
 Often goes undiagnosed for many years and is associated with a strong genetic predisposition, with patients at increased
risk with an increased age (40 years old and above), obesity and lack of physical exercise
 Ketosis seldom occurs but patients are more likely to go into a hyperosmolar coma and are at an increased risk of
developing macrovascular and microvascular complications

GESTATIONAL DIABETES MELLITUS (GDM)

 Any degree of glucose intolerance with onset or first recognition during pregnancy
 Causes of GDM include metabolic and hormonal changes
 Patients with GDM frequently return to normal postpartum

SIGNS AND SYMPTOMS

 Polyuria – excessive urine excretion

 Polydypsia – excessive thirst
 Polyphagia – excessive hunger
 Rapid weight loss (Type 1) or weight gain (Type 2)
 Hyperventilation
 Mental confusion
 Possible loss of consciousness (due to increased glucose in brain)

KETOSIS – accumulation of ketone bodies

Consequences of ketosis

 Metabolic acidosis- Blood pH

 lower than normal (7.4)
 Electrolyte Imbalance
o Decreased Na+ - needed to make ketone bodies soluble
o Increased K+ - fails to be delivered to cells along with glucose
 Necrosis – tissue death; promotes infection particularly of Clostridium perfringens

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GLUCOSE DETERMINATION AND DIAGNOSING DIABETES MELLITUS

BLOOD GLUCOSE LEVELS:
1. NORMOGLYCEMIA – normal glucose levels = 65-100 mg/dL
2. HYPOGLYCEMIA – low blood glucose levels = <65 mg/dL
3. HYPERGLYCEMIA – high blood glucose levels = >100 mg/dL
4. RENAL THRESHOLD – highest level of glucose in blood circulation; glucose appears in urine = 140-160 mg/dL
5. “PANIC” VALUES – dangerously high or dangerously low glucose levels
*too low = <35 mg/dL ~ irreversible brain damagae
*too high = >500 mg/dL ~ multiple organ failure

*BLOOD GLUCOSE PATTERN AFTER MEALS:

160
Glucose levels reaches peak
140
Glucose levels begin to decrease
120
100
Glucose levels begin to rise
80
Normal glucose levels Glucose levels return to normal
60
40
20
0
Before Eating 30 minutes 1 hour 1 hour, 30 minutes 2 hours

MEASUREMENT OF BLOOD GLUCOSE LEVELS:

SPECIMEN COLLECTION:
1) SERUM – most common specimen for quantitative analysis in Clinical Chemistry
– From Red Top (plain) tubes – only for immediate determinations; delay *External Glycolysis Rate:
may cause glycolysis by bacteria and RBC 7%/hr @ Room Temp,
– From Gray Top tubes – with NaF and iodoacetate – glucose preservatives; 2%/hr @ Refrigeration,
prevents glycolysis by inactivating enzymes (enolase and glycerophosphatase, 0%/hr @Freezing Temp
respectively)

Arterial Blood – higher glucose levels than venous blood

– Glucose is still unused by cells (to be delivered)

Venous Blood – lower glucose levels than arterial blood

– Glucose already used up by cells

Capillary Blood – same with arterial blood

– Essentially, capillary blood is still arterial blood

2) CEREBRO-SPINAL FLUID (CSF) – comprises only 65-75% of total serum glucose levels
*Low CSF Glucose Levels (in comparison to serum glucose levels) indicate bacterial meningitis
*Low glucose levels are due to bacterial glycolysis

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3) URINE – specimen used for qualitative analysis in Clinical Microscopy determinations

BLOOD GLUCOSE METHODOLOGY

*PROTEIN FREE FILTRATE (PFF) PREPARATION

Proteins cause precipitation, turbidity, effervescence; must be removed
1) Folin-Wu Method/ Tungstic Acid PFF
Formula: Reaction:
TOTAL VOLUME: 10mL (10% serum)
1 mL serum Serum + [Na2WO4 + H2SO4]  protein ppt
2 mL TypeII RGW H2WO4 (tungstic acid)
2 mL 10% Na2WO4 *responsible for protein precipitation
2 mL 2/3 N H2SO4

× PFF contains blood sugars, not glucose alone

× Saccharoids (sugar-like compounds) are not removed in this method, only proteins

2) Nelson-Somogyi Method/ Zinc Hydroxide PFF

Formula: Reaction:
TOTAL VOLUME: 10mL (10% serum) *responsible for removing saccharoids
BaSO4
1 mL serum
5 mL TypeII RGW Serum + [Ba (OH)2 + ZnSO4]  protein ppt
2 mL 0.3 N Ba(OH)2
Zn (OH)2 (Zinc Hydroxide)
2 mL 5% ZnSO4 *responsible for protein precipitation

 PFF contains blood glucose; allows definite measurement of glucose

 Saccharoids are removed (with the help of BaSO4) along with proteins

3) Van Slyke Method

Formula:
TOTAL VOLUME: 10mL (10% serum) *important measure for iPO4
1 mL serum (inorganic phosphate)
9 mL 10% TCA (Trichloroacetic acid)

*GLUCOSE MEASURE IN CLINICAL SPECIMENS

These methods utilize the reducing property of glucose
1) Copper Reduction Method – glucose reduces Cupric (Cu+2) to Cuprous (Cu+)

a. Folin-Wu Method – makes use of Folin-Wu PFF

Reaction:

+2 Glucose + Cu+ Molybdenum

Cu *reduced
Cu PMA
(Phosphomolybdic Acid) Blue Solution

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× Takes too long to produce results

× Leads to false levels of glucose (due to presence of saccharoids)
b. Nelson-Somogyi Method – makes use of Nelson-Somogyi PFF
Reaction:

+2 Glucose + Cu+ Molybdenum

Cu *reduced Cu AMA
(Arsenomolybdic Acid) Blue Solution
× Takes too long to produce results
× Arsenic (from AMA) is toxic
 Measures true blood glucose levels

2) O-toluidine Method (Dubouski reaction)

Reaction:
∆ (boil)
Glucose + O-toluidine Glycosylamine + Schiff’s base
solution + Glacial acetic acid *green

× Glacial acetic acid is caustic and carcinogenic

3) Glucose Oxidase (GOD) – enzyme that oxidizes glucose

Reaction: *initially colorless
GOD
Glucose + O2 Gluconic Acid + H2O2 *has both oxidizing and
reducing properties
Hydrogen Peroxide is measured through:
4) Trinder’s Reaction
5) Clark’s Method – uses peroxidase; measures O2 by clark electrode
6) O-dianisidene – changes color from colorless to orange brown

7) Hexokinase – enzyme that catalyzes ATP reaction

Reaction:

Hexokinase
Glucose + ATP G-6-P + ADP
G-6-P Dehydrogenase
G-6-P + *colored
NAD NADH + PGA
*colorless (Phosphogluconic acid)

Difference is measured

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8) Autoanalyzer
Principle Involved: Ferricyanide Reaction

Fe (CN) 6-3 Fe (CN) 6-4

*yellow *colorless

Difference is measured
DIAGNOSING DIABETES MELLITUS:

SCREENING TESTS – very SENSITIVE, not too SPECIFIC; measures TRUE POSITIVE values

a.) Fasting Blood Glucose – requires 8-12 hours of Fasting prior to blood collection
*Hyperglycemia suggests Diabetes Mellitus
b.) 2 hour post-prandial – blood is collected two hours after meal
*blood glucose levels return to normal 2 hours after a meal
*elevated glucose levels suggest Diabetes Mellitus

CONFIRMATORY TESTS – SPECIFIC tests for patients with >100mg/dL blood glucose in screening tests

Glucose Tolerance Test (GTT) or Glucose Challenge Test

Patient Preparation:
 150g/day Carbohydrate Diet
 No hard labor/exercise (especially on the day of the test)
i. Oral Glucose Tolerance Test
-administered by Registered Medical Technologist
-non-invasive approach
-Oral Glucose Challenge:
 Adults: 75g glucose
 Pregnant Women: 100 g glucose
 Child (<17y/o): 1.75g glucose per kg of body weight
ii. Intravenous Glucose Tolerance Test
-administered by Registered Nurses
-done in severe cases of hyperglycemia
-more convenient in patients who cannot intake food (i.e. unconscious patients)

National Diabetes Data Group (NDDG) Criteria for diagnosing Diabetes Mellitus:
-up to 140mg/dL on two separate occasions from FBS Screening
-2 out of 3 hyperglycemic results in GTT

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MONITORING TESTS – done during and after patients’ therapeutic regimen prescribed by physician

1) HbA1c – glycated Hemoglobin

*Excessive glucose and Hemoglobin forms glucohemoglobin/glycosylated hemoglobin (unstable)
*Eventually stabilizes to glycated hemoglobin (HbA1c)
* HbA1c is measured through Column Chromatography
* HbA1c is derived from red Blood cells; this test is done once every three months (*RBC Life Span=120 days)
*In Iron Deficiency Anemia, or in other conditions where RBC Life Span is longer, HbA1c levels are higher

2) Fructosamine (FS)
*Glucose and Albumin forms Fructosamine
*FS is measured through Column Chromatography
*This test is done once every three weeks (*Albumin half-life: 21 days)

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