Disclaimer: The notes I’m sharing may not be currently updated (as I’ve used some of them in my past lectures/reviews), so please always refer to textbooks if you encounter conflicting information.
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ALL NOTES MAY BE USED FOR UNDERGRADUATE, LOCAL BOARD EXAM OR INTERNATIONAL EXAMS (i.e., ASCPi)
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ERRATUM FOR THIS:For the action of insulin, please change GLYCOGENOLYSIS to GLYCOGENESIS. Insulin lowers glucose and so it should increase conversion of glucose to glycogen (GLYCOGENESIS).
Compounds containing C, H and O with general formula Cx(H2O)y
Contain C=O and –OH functional groups
Derivatives can be formed by addition of other chemical groups such as phosphates, sulfates and amines
Commonly called “SUGARS” and use the suffix –ose
CLASSIFICATION
Based on four different properties
SIZE OF THE BASE CARBON CHAIN
TRIOSES: with three (3) carbons
TETROSES: with four (4) carbons
PENTOSES: with five (5) carbons
HEXOSES: with six (6) carbons
LOCATION OF THE CO FUNCTION GROUP
ALDOSE: has a terminal carbonyl group (O=CH–) called an aldehyde group
KETOSE: has carbonyl group (O=CH–) in the middle linked to two other carbon atoms called a ketone group
STEREOCHEMISTRY OF THE COMPOUND
STEREOISOMERS: have the same order and types of bonds but different spatial arrangements and different properties
ENANTIOMERS: images that cannot be overlapped and are non-superimposable
L-isomer: if the configuration of the highest-numbered asymmetric carbon is on the LEFT or if hydroxyl group farthest from the carbonyl carbon is on the LEFT
D-isomer: if the configuration of the highest-numbered asymmetric carbon is on the RIGHT or if hydroxyl group farthest from the carbonyl carbon is on the RIGHT
NUMBER OF SUGAR UNITS
MONOSACCHARIDES
Simple sugars that cannot be hydrolyzed to simpler form
Examples: glucose, fructose, galactose
DISACCHARIDES
Formed by two monosaccharides joined by glycosidic linkage
Hydrolyzed by disaccharide enzymes (i.e., lactase) produced by the microvilli of the intestine
Examples:
Maltose = 2 β-D-glucose in 1→4 linkage
Lactose = glucose + galactose
Sucrose = glucose + fructose
OLIGOSACCHARIDES
Chaining of 2 to 10 sugar units
POLYSACCHARIDES
Linkage of many monosaccharide units
Yield more than 10 monosaccharides upon hydrolysis
Examples: starch, glycogen
MODELS USED TO REPRESENT CARBOHYDRATES
FISCHER: linear formula where the aldehyde or ketone is at the top of the drawing and can be depicted in the D- or L- form
HAWORTH: cyclic form that is more representative of the actual structure and is formed when the carbonyl group reacts with an alcohol group on the same sugar to form a ring and can be depicted in the α or β form
The only CHO that can be directly used for energy or stored as glycogen
FORMS: ~35% alpha & 65% beta
MAJOR METABOLIC PATHWAYS
EMBDEN-MEYERHOFF PATHWAY or GLYCOLYSIS
Substrate: D-glucose
End-products: 2 moles of PYRUVIC ACID, 2 moles NADH and 2 moles of ATP
Can occur aerobically or anaerobically
If aerobic, pyruvate is formed
If anaerobic, lactate is formed
Other substrates can enter this pathway at various points
Glycerol (from TAG) enters at 3-phosphoglycerate
Fatty acids, ketones and some amino acids are converted to acetyl-CoA
Other amino acids enter as pyruvates or as deaminated α-ketoacids and α-oxoacids
HEXOSE MONOPHOSPHATE SHUNT OR AEROBIC/OXIDATIVE PATHWAY
G6P is converted to 6-phosphogluconic acid which permits the formation of NADPH (important to red cells because they lack mitochondria thus incapable of TCA cycle)
End-products: pentose phosphate, CO2 and NADPH
GLYCOGENESIS
Stores glucose as glycogen
Converts G6P to G1P
G1P → uridine diphosphoglucose→ glycogen by glycogen synthase
GLYCOGENOLYSIS – conversion of glycogen to G6P
PATHWAYS IN GLUCOSE METABOLISM
Glycolysis
Metabolism of glucose molecule to pyruvate or lactate for production of energy
Gluconeogenesis
Formation of G6P from noncarbohydrate sources
Glycogenolysis
Breakdown of glycogen to glucose for use as energy
Glycogenesis
Conversion of glucose to glycogen for storage
Lipolysis
Decomposition of fats
Lipogenesis
Conversion of carbohydrates to fatty acids
MAJOR HORMONES CONTROLLING BLOOD GLUCOSE
PANCREATIC HORMONES
INSULIN – primary hormone for DECREASING blood glucose levels
Responsible for the entry of glucose into the cells by enhancing membrane permeability to cells in the liver, muscle and adipose tissues
synthesized by β-cells of the pancreas
released when glucose levels are high/increased
not released when glucose levels are low/decreased
EFFECTS:
increases glycogenesis, lipogenesis, and glycolysis
inhibits glycogenolysis
INSULIN IS THE ONLY HORMONE THAT DECREASES GLUCOSE LEVELS and can be referred to as a hypoglycemic agent
GLUCAGON – primary hormone for INCREASING blood glucose levels
released in response to stress and fasting states
synthesized by α-cells of the pancreas
released when glucose levels are low/decreased
not released when glucose levels are high/increased
EFFECTS:
increase glycogenolysis and gluconeogenesis
can be referred to as a hyperglycemic agent
SOMATOSTATIN
produced by δ cells of the pancreas
EFFECTS: inhibition of insulin, glucagon, growth hormone, and other endocrine hormones.
ADRENAL HORMONES
CORTISOL
produced by the adrenal cortex on stimulation by ACTH
EFFECTS: decreases intestinal entry into the cell and increases gluconeogenesis, liver glycogen and lipolysis
EPINEPHRINE
produced by the adrenal medulla
EFFECTS: inhibits insulin secretion, increase glycogenolysis and lipolysis
Released during times of stress
ANTERIOR PITUITARY HORMONES
GROWTH HORMONE
EFFECTS: decreases the entry of glucose into the cells
ACTH
EFFECTS: stimulates the adrenal cortex to release cortisol, increases glycogenolysis and gluconeogenesis
THYROID HORMONES
T3 & T4
EFFECTS: increases glycogenolysis, gluconeogenesis and intestinal absorption of glucose
glycogenolysis, gluconeogenesis & intestinal absorption of glucose
Somatostatin
δ cells of pancreas
↑
inhibits insulin, glucagon, GH
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Hyperglycemia
Increase in plasma glucose levels caused by imbalance of hormones
DIABETES MELLITUS
Group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both
Categories of Diabetes (According to the ADA/WHO guidelines)
Type 1 Diabetes
Type 2 Diabetes
Other specific types of diabetes
Gestational Diabetes Mellitus (GDM)
PRIMARY DIABETES MELLITUS
Points of Difference
TYPE 1
TYPE 2
Former names
Insulin Dependent Diabetes Mellitus (IDDM)
Juvenile Onset DM
Brittle DM
Ketosis-prone DM
Non-Insulin Dependent Diabetes (NIDDM)
Maturity Onset DM
Stable DM
Ketosis-resistant DM
Receptor Deficient DM
Onset
Before 20 y/o
Over 40 y/o
Measurable circulating insulin
NONE
Low
Insulin receptor
Normal
↓ or ineffective
Beta cell mass
Markedly ↓
Moderately ↓
C-peptide levels
Undetectable
Detectable
Incidence
10-15%
85% (common)
Ketoacidosis*
Common
Rare
Physique/Stature**
Normal or thin
Often overweight
Pathogenesis
-β-cell destruction
-Absolute insulin deficiency
-Autoantibodies
-Insulin resistance with insulin secretory defect
-Relative insulin deficiency
Treatment
Parenteral insulin administraion
Oral hypoglycemic agent
SECONDARY DIABETES MELLITUS – associated with secondary conditions
Genetic defects of β-cell function
Pancreatic disease
Endocrine disease
Cushing syndrome – excessive cortisol
Pheochromocytoma – epinephrine excess
Acromegaly – growth hormone excess
Drug or chemical induced
Insulin receptor abnormalities
Other genetic syndromes
Maturity onset diabetes of youth (MODY) – rare; autosomal dominant
GESTATIONAL DIABETES MELLITUS (GDM)
any degree of glucose intolerance with onset or first recognition during pregnancy
due to metabolic or hormonal changes
Infants born to mothers with this kind of diabetes are at increased risk to respiratory distress syndrome, hypocalcemia & hyperbilirubinemia
Laboratory Findings in Hyperglycemia
INCREASED glucose (plasma & urine), urine specific gravity, serum and urine osmolality
Ketonemia and ketonuria
DECREASED blood and urine pH (acidosis)
Electrolyte imbalance (↓Na+, Cl– and ↑K+)
DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS
RPG ≥200 mg/dl (11.1 mmol/L) + symptoms of diabetes
Fasting PG ≥126 mg/dL (7.0 mmol/L)
2-h PG ≥200 mg/dl (11.1 mmol/L) during OGTT
CATEGORIES OF FASTING PLASMA GLUCOSE
Normal fasting glucose FPG <110 mg/dL
IMPAIRED fasting glucose FPG ≥110 mg/dl but <126 mg/dl
Provisional diabetes dx FPG ≥126 mg/dl
CATEGORIES OF ORAL GLUCOSE TOLERANCE
Normal glucose tolerance 2h PG <140 mg/dL
Impaired gluc. tolerance 2h PG ≥140 mg/dl but <200 mg/dl
Provisional diabetes dx 2h PG ≥200 mg/dl
Screening test for GDM
Only high-risk patients should be screened for GDM
Age older than 25 years
Overweight
Strong family history of diabetes
History of abnormal glucose metabolism
History of a poor obstetric outcome
Presence of glycosuria
Diagnosis of PCOS
Member of an ethnic/racial group with a high prevalence of diabetes (e.g. Hispanic American, Native American, Asian American, African American, Pacific Islander)
METHODS:
ONE-STEP APPROACH – immediate performance of a 3h OGTT without prior screening
TWO-STEP APPROACH – initial measurement of plasma glucose at 1-hour postload (50g)
IF value ≥140 mg/dL (7.8 mmol/L) then do 3-hour OGTT using 100g glucose
GDM is diagnosed when any two of the following values are met or exceeded:
Fasting: >95 mg/dl
1 hour: ≥180 mg/dl
2 hours: ≥155 mg/dl
3 hours: ≥140 mg/dl
Hypoglycemia
Decrease in plasma glucose levels
65-70 mg/dl (3.6-3.9 mmol/L) – plasma glucose concentration at which glucagon and other glycemic factors are released
50-55 mg/dl (2.8-3.0 mmol/L) – symptoms of hypoglycemia appear
Warning S/S are all related to CNS
Types of Hypoglycemia (Old)
Post-absorptive (Fasting) – MORE SERIOUS
Islet cell insulinoma
Insulin-producing tumors
Ethanol induced
Propanolol & salicylate
Post-prandial (Reactive) – MILD FORM
there is spontaneous recovery of glucose level as a result of insulin level returning to normal
Excessive release of insulin
Gastro-intestinal surgery
CAUSES OF HYPOGLYCEMIA
Patient Appears Healthy
No coexisting disease
Drugs
Insulinoma
Islet hyperplasia or NESIDIOBLASTOSIS
Factitial hypoglycemia from insulin or sulfonylurea
Severe exercise
Ketotic hypoglycemia
Compensated coexistent disease
Drugs
Patient Appears ILL
Drugs
Predisposing illness
Hospitalized patient
Diagnostic criteria for INSULINOMA
Change in glucose level of ≥25 mg/dl coincident with an insulin level of ≥6 μU/ml
C-peptide levels of ≥0.2 nmol/L
Proinsulin levels of ≥5 pmol/L
β-hydroxybutyric acid of ≤2.7 mmol/L
Diagnostic tests for HYPOGLYCEMIA
72 hour fast which requires the analysis of glucose, insulin, C-peptide and proinsulin at 6-hour intervals
POSITIVE RESULT: <45 mg/dl; hypoglycemic symptoms appear after 72 hours had elapsed
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Genetic Defects in Carbohydrate Metabolism
Glycogen Storage Diseases – deficiency of a specific enzyme that causes alteration of glycogen metabolism
Types
Enzyme Deficient
Clinical Features
von Gierke’s dse
Type I
Glucose-6-phosphatase
Severe fasting hypoglycemia
Lactic acidosis
Pompe’s dse
Type II
α-1,4-glucosidase
Accumulation of ↑ amount of glycogen on all organs
Presence of abnormally LARGE LYSOSOMES
Forbe’s dse
Type III
Debrancher enzyme
Hypoglycemia, hepatomegaly, seizures and mental retardation
Andersen’s dse
Type IV
Brancher enzyme
Progressive liver enlargement or cirrhosis and muscular weakness by age 2
Absence of storage glycogen
Unbranched AMYLOPECTIN
Other enzyme defects/deficiencies that cause hypoglycemia: glycogen synthase, fructose-1-6,biphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase.
Galactosemia – a cause of failure to thrive syndrome in infants; congenital deficiency of one of three enzymes involved in galactose metabolism, resulting in increased plasma galactose levels
Galactose-1-phosphate uridyl transferase – MOST COMMON enzyme deficiency
Fructose-1-phosphate aldolase deficiency
Laboratory Analysis of Glucose
SPECIMEN COLLECTION AND HANDLING
Glucose concentration in whole blood is approximately 15% lower than in plasma or serum.
Glucose levels decrease approximately 10 mg/dL (7%) per hour in whole blood.
Serum or plasma must be separated within 1 hour (Bishop) to prevent substantial loss of glucose by the cellular fraction, particularly if WBC count is elevated. (within 30 minutes – Henry)
Glucose is metabolized at a rate of 7 mg/dl/h at room temperature; and 2 mg/dl/h at 4°C
Refrigerated serum or plasma is stable up to 48 hours.
Sodium fluoride (2 mg/mL) prevents glycolysis (gray top tube) for up to 48 hours.
Glycolysis decrease serum glucose by approximately 5-7% per hour (5-10 mg/dl) in normal, uncentrifuged coagulated blood at room temperature.
Fasting blood glucose should be obtained after an approximately 10-hour fast (not >16 hours)
Fasting plasma glucose values have a diurnal variation with the mean FBG higher in the morning than in the afternoon.
Fasting reference range for serum or plasma is 70-110 mg/dL
In the fasting state, arterial (capillary) values are 5 mg/dL higher than the venous concentration.
Urine glucose analysis (in 24h urine glucose) may be stabilized by addition of a preservative; should be stored at 4°C during collection because 40% of glucose is lost after 24 hours at room temperature.
CSF glucose analysis (if will be delayed) must be centrifuged and stored at 4°C-20°C
In normal CSF, values are two-thirds (approximately 60-70%) of plasma level.
RENAL THRESHOLD for glucose: 180 mg/dl
TYPES OF SPECIMEN FOR GLUCOSE ANALYSIS
Fasting Blood Sugar – blood collected after 8-10 hours of fasting (NV: 74-106 mg/dl)
Random Blood Sugar – test for INSULIN SHOCK (NV: <200 mg/dl)
2 hour Postprandial Blood Sugar
Standard load of glucose: 75 grams
Glucose measurement taken 2 hours later
(NV : <120 mg/dl)
Glucose Tolerance Test – multiple blood and urine glucose test
Oral GTT
Janney-Isaacson (Single Dose)
Exton Rose (Divided Oral dose or Double Dose)
Not recommended for routine use
Fasting and 2h sample are measured except for pregnant patients
Adult load is 75g; children: 1.75 g/kg to 75g
Factors that affect tolerance
Medications (salicylates, diuretics, anticonvulsants, oral contraceptives and corticosteroids)
GI surgery
Vomiting
Endocrine dysfunction
Requirements:
Patient should be ambulatory
Patient must be in unrestricted diet of 150 grams CHO/day for 3 consecutive days prior to the test
Patient must be free from undue stress or severe illness
Alcohol intake and smoking are not allowed prior to the test
Patient should be fasting at least 10 hours and not more than 16 hours
Test should be performed in the morning because of hormonal diurnal effect on glucose
IVGTT – blood sample is collected every 10 minutes for 1 hour
5g glucose/kg body weight (given within 3 minutes) administered intravenously
fasting is also required
NV: 1.4 – 2.0 %
Indications of IVGTT
Patients who are unable to tolerate large CHO load
Patients with altered gastric physiology or GI d/o
Patients with malabsorption syndrome
Self-Monitoring of Blood Glucose (SMBG)
Type 1 DM – should monitor blood glucose 3-4 times per day
Type 2 DM – optimal frequency is unknown
Glycosylated hemoglobin/Glycated hemoglobin/HbA1C
hemoglobin compound formed when glucose reacts with amino group of hemoglobin
test for long term diabetic control
reflects the average blood glucose level for the previous 2-3 months
for every 1% change in HbA1c value there is 35 mg/dl (2 mmol/L) change in the mean
in presence of hemoglobinopathies, there will be less time for glucose to
binding of glucose to HbA1 is irreversible
preferred anticoagulant is EDTA
NV: 4.5-8.5%
Methods of HBA1c Measurement
Methods based on STRUCTURAL DIFFERENCES
Immunoassays
Polyclonal or monoclonal antibodies toward the glycated n-terminal group of the β chain of Hgb
Affinity chromatography
Separates based on chemical structure using borate to bind glycosylated proteins
Not affected by temperature and other hemoglobins
Methods based on CHARGE DIFFERENCES
Ion-exhange chromatography
Positive-charge resin bed
Highly affected by temperature and hemoglobinopathies
HbF – ↑
HbS and C – ↓
Electrophoresis
Separation is based on differences in charge
HbF values >7% interferes
Isoelectric focusing
Type of electrophoresis using isoelectric point to separate
Pre-hb A1c interferes
HPLC
Form of ion-exchange chromatography
Separates all forms of glycol Hb (a,b,c)
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METHODS FOR ANALYSIS
CHEMICAL
REDUCTION
Cupric Ion Reduction
FOLIN-WU – measure of ALL REDUCING SUBSTANCES in the blood
Reagent that binds with Cu+: phosphomolybdic acid
End product: phosphomolybdenum blue
End color: blue
NELSON SOMOGYI – MEASURE OF TRUE GLUCOSE
Reagent that binds with Cu+: arsenomolybdic acid
End product: arsenomolybdenum blue
End color: blue
NEOCUPROINE
Reagent that binds with Cu+: neocuproine
End product: cuprous-neocuproine complex
End color: yellow/yellow orange
Ferric Ion Reduction – Inverse Colorimetry – reduction of yellow ferricyanide to a colorless ferrocyanide by glucose
HAGEDORN JENSEN
CONDENSATION
Orthotoluidine (DUBOWSKI method)
can be also used for urine and CSF without protein precipitation
Absorbance: 630 nm
Reagent: aromatic amine, glacial acetic acid
End color: green
Interfering substances: galactose and mannose
Polarographic Glucose Oxidase
measures oxygen consumption with PO2 electrode (Clark)
used to avoid interference made by strong oxidizing agents in GOD
Molybdate – catalyzes the oxidation of iodide to iodine by H2O2
Catalase – catalyzes oxidation of ethanol by H2O2 forming acetaldehyde and H2O
Hexokinase
Generally accepted as the REFERENCE METHOD
MORE ACCURATE THAN HEXOKINASE
coupling reaction using G6PD is highly specific
Measured by quantitating reduced NADPH formation
NADPH is measured directly at 340 nm or coupled to chromogen and measured in visible range
May be performed using serum or plasma (heparin, EDTA, fluoride, oxalate & citrate)
Excellent for glucose determination in urine, CSF and serous fluids
OTHER IMPORTANT TESTS
KETONES
Produced by the liver through metabolism of fatty acids to provide ready energy source from stored lipids at times of low carbohydrate availability
THREE KETONE BODIES
Acetone (2%)
Acetoacetic acid (20%)
Β-hydroxybutyric acid (78%)
Causes of increased ketone levels
Diabetes Mellitus
Starvation/fasting
High-fat diets
Prolonged vomiting
Glycogen storage diseases
KETONEMIA – accumulation of ketones in the blood
KETONURIA – accumulation of ketones in the urine
MEASUREMENT OF KETONES
For patients with Type 1 Diabetes, it is recommended during acute illness, stress, pregnancy, or elevated blood glucose levels above 300 mg/dL or when patients have signs of ketoacidosis
SPECIMEN: FRESH SERUM or URINE tightly stoppered and analyzed immediately
METHODS FOR ANALYSIS:
GERHARDT’S TEST – historical test
Used FERRIC CHLORIDE reacted with ACETOACETIC ACID to produce a RED color
SODIUM NITROPRUSSIDE – more common method
Uses SODIUM NITROPRUSSIDE which reacts with ACETOACETIC ACID in an ALKALINE pH to form a PURPLE COLOR
If GLYCERIN is also added, ACETONE will be detected
Used in urine reagent strips and Acetest tablets
ENZYMATIC – newer method adapted in some automated intstruments
Uses β-HYDROXYBUTYRATE DEHYDROGENASE to detect either β-HYDROXYBUTYRIC ACID or ACETOACETIC ACID depending on the pH of the solution
pH of 7.0 causes the reaction to proceed to the right (decreasing absorbance)
pH of 8.5 to 9.5 causes the reaction to proceed to the left (increasing absorbance)
MICROALBUMINURIA
Defined as persistent albuminuria in the range of 30 to 299 mg/24 h or an albumin-creatinine ratio of 30 to 300 g/mg
Clinical proteinuria or macroalbuminura is established with an albumin-creatinine ratio of ≥300 mg/24h or ≥300 µg/mg
Powerful predictor for future development of diabetic nephropathy
Annual assessment of kidney function by the determination of urinary albumin is recommended for diabetic patients
METHODS FOR MICROALBUMINURIA SCREENING
RANDOM SPOT TEST – preferred method
24-HOUR COLLECTION
TIMED 4-HOUR OVERNIGHT COLLECTION
A patient is determined to have microalbuminuria when two of three specimens collected within a 3- to 6-month period are abnormal.
Factors that may elevate the urinary excretion of albumin include exercise within 24 hours, infection, fever, congestive heart failure, marked hyperglycemia, and marked hypertension
ISLET AUTOANTIBODY AND INSULIN TESTING
Not currently recommended for routine screening for diabetes diagnosis but in the future it might identify at-risk, prediabetic patients
TESTS FOR CARBOHYDRATE DISORDERS
DIAGNOSTIC TESTS
ACTION
Fasting Blood Sugar
Normal – 70-110 mg/dl
Diabetes – >126 mg/dl
2hr Post Prandial Blood Sugar (PPBS)
Normal – <126 mg/dl
Diabetes – >200 mg/dl
Post-Loading Glucose
Similar to PPBS
*Glucose load is standardized
*Diabetics ≥200 mg/dl
Glucose Tolerance Test (GTT) Standard dose = 75g
*Diagnostics of diabetes mellitus
>150 mg/dl after 2 hours
>200 mg/dl after 2 hours
*Perform if FBS and PPBS are normal
Intravenouse Glucose Tolerance Test (IVGTT)
*Poor absorption (flat curve with OGTT)
*Patient who cannot tolerate large glucose load (vomiting)
O’Sullivan Test
(for gestational diabetes)
*Standard dose 50g
*Probable gestational diabetes
>150 mg/dl at 1 hour
*Follow up with OGTT
TESTS FOR MONITORING
NOTES
Glycosylated hemoglobin
*Assessment of long term control
*Average glucose level over 60 days (1-2 months)
Microalbumin
*Detects small amounts of protein in urine of diabetic patients to assess renal damage