WHAT’S IN HERE?

• Carbohydrates
• Glucose and Its Metabolism
• Hyperglycemia
• Hypoglycemia
• Genetic Defects in Carbohydrate Metabolism
• Laboratory Analysis of Glucose
• References

DEFINITION

• Compounds containing C, H and O with general formula C_x(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

CHEMICAL PROPERTIES

• REDUCING SUBSTANCES
  • Contain a ketone or aldehyde group
  • WITH FREE ANOMERIC CARBON
  • Can reduce other compounds
  • Examples: glucose, maltose, fructose, lactose, galactose
• NON-REDUCING SUBSTANCES
  • Do not have an active ketone or aldehyde group
  • NO FREE ANOMERIC CARBON
  • Will not reduce other compounds
  • Example: sucrose (table sugar)

Glucose and Its Metabolism

• End product of carbohydrate digestion in the intestine
  • Enzymes involved
    • AMYLASE (salivary & pancreatic) – digests nonabsorbable polymers to dextrins and disaccharides
    • MALTASE (from the intestine) – digests disaccharides to monosaccharides
    • SUCRASE & LACTASE – hydrolyze sucrose & lactose respectively
  • FUNCTIONS:
    • Provides energy for life processes
    • 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

• 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

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

• 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

• 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

• 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

Genetic Defects in Carbohydrate Metabolism

• Glycogen Storage Diseases – deficiency of a specific enzyme that causes alteration of glycogen metabolism

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 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
    • Interfering substances: gross hemolysis & extremely elevated bilirubin (cause ↓ values)
    • 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

REFERENCES:

1. Bishop, Michael L., et.al., Clinical Chemistry Techniques, Principles, Correlations, Sixth Edition
2. PER Handbook
3. Theriot, Betty, Clinical Laboratory Science Review: Bottom Line Approach
4. McPherson, Richard, et.al., Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22e