Carbohydrates

- Macronutrients in our diet

- Function: provide energy in the body
- Different forms: sugars and dietary fibers
- Whole grains, fruits, vegetables
- Carbohydrates broken down to glucose is the preferred energy source

A. Pathways in Glucose Metabolism

Glycolysis – metabolism of glucose molecule to pyruvate or lactate for production of energy; blood
glucose is decreased

Gluconeogenesis – formation of glucose-6-phosphate from noncarbohydrate sources (increases blood

glucose)

Glycogenolysis – breakdown of glycogen to glucose for use as energy (blood glucose is increased)

Glycogenesis – conversion of glucose to glycogen for storage (blood glucose is decreased)

Lipogenesis – conversion of carbohydrates to fatty acids (blood glucose is decreased)

Lipolysis – decomposition of fat (Blood glucose is increased)

Regulation of Blood Glucose

Terminologies:

Hyperglycemia – elevated blood glucose

Hypoglycemia – low blood glucose

The two main hormones that control blood glucose are: insulin (beta-cells of pancreas) and glucagon
(alpha cells of pancreas)

High blood glucose (Hyperglycemia)

- Insulin released by the pancreas

- Insulin stimulates cells to take up glucose from the blood
- Insulin stimulates liver and muscle cells to store glucose as glycogen

Low blood glucose (Hypoglycemia)

- Glucagon released from pancreas

- Glucagon stimulates liver cells to break down glycogen to glucose
- Glucagon stimulates liver cells to make glucose from amino acids
Hormones that regulate blood sugar level:

A. Hormones that increase blood glucose

- Glucagon – promotes gluconeogenesis and glycogenolysis
- ACTH – stimulates the adrenal cortex to release cortisol; glycogenolysis and gluconeogenesis
- Growth Hormone – glycogenolysis and glycolysis
- Cortisol – decreases intestinal entry into the cell; gluconeogenesis and lipolysis
- Human Placental Lactogen – promotes breakdown of maternal fats (lipolysis); causes maternal
diabetes
- Epinephrine – inhibits insulin secretion; glycogenolysis and lipolysis
- T4 (Thyroxine) – intestinal absorption of insulin; glycogenolysis and gluconeogenesis
- Somatostatin – inhibits insulin, glucagon, growth hormone, and other endocrine hormones
B. Hormones that decrease blood glucose
- Insulin – primary hormone responsible for the entry of glucose into the cell; also regulates
glucose by increasing glycogenesis, lipogenesis and glycolysis, and inhibiting glycogenolysis

LABORATORY METHODS

Glucose Measurements

Specimen considerations:

- Whole blood, plasma, serum, CSF, pleural fluid, and urine

- Most glucose measurements are performed on serum or plasma
- Capillary blood for point of care testing (POCT), via capillary puncture
- Venous plasma glucose is the standard clinical specimen; blood is obtained via venipuncture,
using anticoagulated tube (gray top tube)
- The glucose concentration in whole blood is approximately 11% lower than serum or plasma
- Glucose is metabolized at room temperature at a rate of 7 mg/dL/hour (0.4 mmol/L/hour); at 4
degrees Celsius, glucose decreases by approximately 2 mg/dL/hour (CONVERSION FACTOR:
0.555 mg/dL  mmol/L)
- A serum specimen is appropriate for glucose analysis if serum is separated from the cells within
30 minutes; if longer than 30 minutes, add sodium fluoride, which inhibits glycolysis (additive
present in gray top tube)
- If whole blood is refrigerated, 2 mg of sodium fluoride per milliliter of whole blood prevents
glycolysis for up to 48 hours; decreasing the blood glucose concentration
- Fasting Blood Sugar (FBS) or Fasting Plasma Glucose (FPG) should be obtained in the morning
after an approximately 8 to 10 hours of fasting (not longer than 16 hours)
- FBS/FPG values have a diurnal variation (FPG higher in the morning than in the afternoon)
- No to hemolysis

SAMPLES FOR GLUCOSE MEASUREMENT

1. Fasting Blood Sugar (FBS) or Fasting Blood Glucose

- Screening test for diabetes mellitus
- Taken after at least 8 hours of fasting
- Usually done in the morning to prevent diurnal variation caused by hormones
- Measure overall sugar homeostasis

Categories of Fasting Plasma Glucose

Normal fasting glucose FPG 70-99 mg/dL
(3.9-5.5 mmol/L)
Impaired fasting glucose FPG 100-125 mg/dL
(not a clinical entity but a risk factor for (5.6-6.9 mmol/L)
diabetes and cardiovascular disease)
Provisional diabetes diagnosis FPG >= 126 mg/dL
- must be confirmed (>= 7.0 mmol/L)
2. Random Blood Sugar
- Taken any time of the day without fasting
- Often used for emergency cases
3. 2-Hour Post Prandial Blood Sugar (PPBS)
- Taken 2 hours after meal
- Measures how well the body metabolizes glucose
4. Glucose Tolerance Test
- Multiple blood sugar test
- Measures how well the body metabolizes glucose over a required period of time
- Used to diagnose gestational diabetes
- Not recommended as routine diagnosis of diabetes
 Oral Glucose Tolerance Test
 Intravenous Glucose Tolerance Test
 Used for patients who are unable to tolerate a large oral carbohydrate load or
may have altered gastric physiology
 The dose of glucose is 0.5 g/kg of body weight
 Administered intravenously

Guidelines for Oral Glucose Tolerance Test (OGTT)

- Patient is asked to consume 150 g of carbohydrate per day for 3 days prior to the test
- Discontinue, if possible, medications known to affect glucose tolerance
- Fast overnight (8 to 10 hours, not greater than 16 hours) and avoid excessive physical
activity
- Perform OGTT in the morning (between 7 am to 9 am) to prevent hormonal diurnal effect
on glucose
- Patient should be ambulatory
- Fasting blood glucose is measured before the glucose load
- FBG > 140 mg/dL: test should be TERMINATED
- FBG < 140 mg/dL: glucose load should be given to the patient
- For OGTT, the glucose load for adult is 75 g; for children, 1.75 g/kg body weight (up to a 75 g
maximum is given)
- The glucose should be dissolved in 300 mL of water, and ingested within 5 minutes
- The patient should not vomit. If the patient vomits, discontinue the test
 CATEGORIES OF ORAL GLUCOSE TOLERANCE
Normal glucose tolerance 2-hour Plasma Glucose =< 140 mg/dL
(=< 7.8 mmol/L)
Impaired glucose tolerance 2-hour Plasma Glucose = 140-199 mg/dL
(not a clinical entity but a risk factor for (7.8-11.1 mmol/L)
diabetes and cardiovascular disease)
Provisional diabetes diagnosis 2-hour Plasma Glucose >= 200 mg/dL
(>= 11.1 mmol/L)

5. Glycosylated Hemoglobin/ Hemoglobin A1c (HbA1c)

- Glycated hemoglobin
- Largest subfraction of normal hemoglobin A in both diabetic and non-diabetic individuals
- More reliable method of monitoring long-term diabetes control over the previous 2-4
month period
- For every 1% increase in HbA1c, there is a 35mg/dL (2 mmol/L) change in plasma glucose
- Results are affected by the average glucose concentration and life span of RBCs (lower RBC
life span, lower HbA1c)
- Specimen of choice: Whole blood in EDTA (purple top tube)

HbA1c results
Normal 4-6%
Increased risk for diabetes 5.7%-6.4%
Diabetes >= 6.5%
6. Fructosamine
- Also known as glycosylated albumin, glycated albumin, plasma protein ketoamine
- Used for monitoring glucose control over the previous 2-3 weeks (20 days = life span of
albumin in the circulation)
- Affected by changes in albumin levels
- Reference value: 205-285 umol/L

METHODS FOR GLUCOSE DETERMINATION

I. Chemical Methods
A. Oxidation Reduction Method
1. Alkaline Copper Reduction Method
Principle: Reduction of cupric ions to cuprous ions forming cuprous oxide in hot
alkaline solution by glucose

Alkaline Copper Tartate glucose Cuprous ions


heat
a. Folin Wu Method

Cuprous ions +  Phosphomolybdic Acid or

Phosphomolybdate Phosphomolybdenum Blue
 b. Nelson Somogyi Method

Cuprous ions +  Arsenomolybdic Acid or

Arsenomolybdate Arsenomolybdenum Blue

c. Neocuprein Method (2,9-Dimethyl-1,10-Phenantroline Hydrochloride)

Cuprous ions +  Cuprous-Neocuprein

Neocuprein Complex (Yellow or Yellow
Orange)

d. Benedict’s Method (Modification of Folin Wu)

- used for the detection and quantitation of reducing substances in body fluids
like blood and urine
- uses citrate or tartate as stabilizing agent

Copper sulfate (blue) +  Brick red precipitate

glucose + heat

2. Alkaline Ferric Reduction Method (Hagedorn Jensen)

- involves reduction of a yellow ferricyanide to a colorless ferrocyanide by glucose
- inverse colorimetry

B. Condensation Method

1. Ortho-toluidine (Dubowski Method)

Glucose + Aromatic Glacial HAC Glycosylamine + Schiff’s

Amines  base (green)
Heat

II. Enzymatic Methods

3 Enzyme systems are commonly used to measure glucose:
- Glucose dehydrogenase
- Glucose oxidase
- Hexokinase

* these enzymes act on glucose but not on other sugars and not on other reducing substances

A. Glucose Dehydrogenae Method

- Glucose is reduced to produce a chromophore that is measure spectrophotometrically
- The amount of NADH generated is directly proportional to the glucose concentration

Alpha-D-glucose  Beta-D-glucose
 (mutarotase)
Beta-D-glucose  D-gluconolactone + NADH
(glucose
dehydrogenase)
(mutarotase) MTT +  MTTH (blue color) + NAD
NADH (diaphorase)
B. Glucose Oxidase method
a. Colorimetric Glucose Oxidase method
- Also known as Saifer Gernstenfield method

Glucose + O2 + H2O  Gluconic acid + H2O2

(glucose oxidase)
H2O2 + reduced  Oxidized chromogen + H2O
chromogen (peroxidase)
Glucose oxidase measures Beta-D-glucose and measures CSF in urine samples

b. Polarographic Glucose Oxidase

- Measures the rate of oxygen consumption which is proportional to glucose concentration

Glucose + O2  Gluconic acid + H2O2

(glucose oxidase)
H2O2 (hydrogen  CH3CHO + 2H2O
peroxide) + C2H5OH (catalase) (acetaldehyde)
(ethanol)
H2O2 (hydrogen  I2 + 2H2O
peroxide) + 2H + 2I (molybdate)
c. Hexokinase Method
- Most specific glucose method; reference method

Glucose + ATP  Glucose 6-PO4 + ADP

(hexokinase)
Glucose 6-PO4 + NADP  NADPH + H + 6-
(G6PD) phosphogluconate

d. Dextrostics (cellular strip)

e. Interstitial glucose measuring devices – for continuous measuring of glucose levels of
diabetic patients

C. CLINICAL SIGNIFICANCE

Hyperglycemia – increase in plasma glucose levels; caused by an imbalance of hormones; diabetes

mellitus

Diabetes Mellitus – group of metabolic diseases characterized by hyperglycemia resulting from defects
in insulin secretion, insulin action, or both

Categories of diabetes:

- Type 1
 - Type 2
- Other specific types of diabetes
- Gestational diabetes mellitus (GDM)

Type 1 diabetes

- Insulin Dependent DM, Juvenile Onset DM, Brittle Diabetes, Ketosis, Prone Diabetes
- Result of cellular mediated autoimmune destruction of the beta-cell of the pancreas
a. Islet cell autoantibodies
b. Insulin autoantibodies
c. Glutamic acid decarboxylase autoantibodies
d. Tyrosine phosphate IA-1 and IA-2B autoantibodies
- Constitutes only 10% to 20% of all cases of diabetes
- Signs and symptoms
a. Polydipsia (excessive thirst)
b. Polyphagia (increased food intake)
c. Polyuria (excessive urine production)
d. Rapid weight loss
e. Hyperventilation
f. Mental confusion
g. Possible loss of consciousness (due to increased glucose in brain)
- Complications: microvascular problems (retinopathy, necropathy, neuropathy)
- Parenteral administration of insulin

Idiopathic type 1 diabetes

- Form of type 1 diabetes that has no known etiology

- Strongly inherited, and does not have beta-cell autoimmunity
- Individuals with this form of diabetes have episodic requirements for insulin replacement

Type 2 diabetes

- Non-insulin dependent DM, Maturity onset or adult type DM, Stable Diabetes, Ketosis-
Resistant Diabetes, Receptor-Deficient DM
- Result of an individual’s resistance to insulin
- Constitutes the majority of the diabetes cases
- Complications: macrovascular and microvascular
- Uses hypoglycemic agents to regulate blood sugar level
- Untreated type 2  nonketotic hyperosmolar coma due to overproduction of glucose
(>300mg/dL); severe dehydration; increased BUN and creatinine
- Risk factors for type 2 DM:
a. Genetic, obesity, sedentary lifestyle, race/ethnicity, PCOS, dyslipidemia,
hypertension

Gestational diabetes mellitus (GDM)

- Has been defined as any degree of glucose intolerance with onset or first recognition during
pregnancy
 - Screening should be performed 24 and 28 weeks of gestation

Other specific types of diabetes

a. Genetic defects of beta-cell function

b. Pancreatic disease
c. Endocrine disease
d. Drug or chemical induced
e. Insulin receptor abnormalities
f. Other genetic syndromes

Lab findings in hyperglycemia:

1. increased glucose in plasma and urine

2. increased urine specific gravity

3. ketones in serum and urine (ketonemia and ketouria)

4. decreased blood and urine Ph (ACIDOSIS)

4. Electrolyte imbalance

DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS

1. HbA1c >= 6.5% using a method that is NGSP certified and standardized to the DCCT assay
2. Fasting plasma glucose >= 126 mg/dL (>=7.0 mmol/L)
3. 2-hour plasma glucose >= 200 mg/dL (>=11.1 mmol/L) during an PGTT
4. Random plasma glucose >= 200 mg/Dl (>=11.1 mmol/L) plus symptoms of diabetes

Hypoglycemia

- Decreased plasma glucose levels

- Glucagon and other glycemic factors are released when plasma glucose concentration is
between 65 and 70 mg/dL (3.6 to 3.9 mmol/L)
- Observable symptoms of hypoglycemia appear at about 50 to 55 mg/dL (2.8 to 3.1 mmol/L)

Symptoms of hypoglycemia:

- Increased hunger
- Sweating
- Nausea and vomiting
- Dizziness
- Nervousness and shaking
- Blurring of sight
- Mental confusion

Laboratory findings:

- Decreased plasma glucose levels

- Extremely elevated insulin levels in patients with pancreatic cell tumors (insulinoma)
INBORN ERRORS OF CARBOHYDRATE METABOLISM

Glycogen Storage Diseases

- Result of the deficiency of a specific enzyme that causes an alternation of glycogen

metabolism
- Most common: von Gierke disease
- The liver forms (types I, III, IV, VI, IX, and 0) are marked by hepatomegaly and hypoglycemia
- muscle forms (types V and VII) are characterized by muscle cramps, exercise intolerance,
fatigue, and weakness

defects in galactose metabolism:

Galactosemia

- cause of failure to thrive syndrome in infants

- resulting in increased levels of galactose in plasma
- enzymes:
a. galactose-1-phosphate uridyltransferase (most common)
b. galactokinase
c. uridine diphosphate galactose-4-epimerase

Defects in fructose metabolism

Essential fructosuria – fructokinase deficiency

Hereditary fructose intolerance – defect in fructose-1,6-biphosphate aldolase B activity

Fructose-1,6-Biphosphatase deficiency – defect in fructose-1,6-biphosphatase

GSD ENZYME DEFICIENT (Tissue Affected)