19 June 2016

Lecture #7: Carbohydrates


Carbohydrates are the major food supply and energy source of the body. Depending on dietary habits, 50 – 90% consumed come from grain, starchy vegetables and legumes. Typical items in this group include rice, wheat, corn and potatoes.

Carbohydrate is the element in our food which supplies the energy for the body’s automatic activity and for the performance of our daily tastes. Besides their usefulness in supplying energy, carbohydrates play a vital part in the digestion, assimilation and oxidation of protein and fat.

Despite the major utilization of carbohydrate for energy, only a small amount of it is stored in the body. The average adult reserve is about 370 grams stored chiefly as liver and muscle glycogen. Since one gram of carbohydrate supplies 4 calories or approximately half the average daily calorie needs. When the total calorie intake exceeds the daily expenditure, the excess carbohydrate is readily converted to fat and stored at adipose tissue.

The most common disease related to carbohydrate metabolism is diabetes mellitus, which is characterized by inability of the body to burn up (assimilate) glucose due to deficient levels of active insulin. Deficiency of insulin results in impaired metabolism, and increases in blood glucose concentration and secondary changes in fat metabolism, leading eventually to ketosis and possible diabetic coma. Other complications include hypercholesterolemia, atherosclerosis and kidney disease. The condition of diabetes shows strong familial tendency; the probability that an individual will develop diabetes is several times greater when there is a family history of disease.

Early recognition of diabetes will permit earlier management and perhaps delay or minimize the complications of the disease. Overproduction or excess administration of insulin causes a decrease in blood glucose level below normal. In severe cases, the resulting extreme hypoglycemia is followed by muscular spasms and loss of consciousness, known as insulin shock. Measurement of blood glucose, therefore, assumes considerable significance and the clinical laboratory must be prepared to furnish results rapidly and with high degree of reliability.

The determination of blood glucose is the procedure most frequently done in hospital chemistry laboratory. In addition, many glucose determinations are performed in clinics, independent laboratories and physician’s office as an aid in the diagnosis and treatment of diabetes. The determination may be performed on patients in the fasting state, in the post prandial state or in conjunction with glucose tolerance test, in accordance with the physician’s request.

The normal glucose concentration in blood is between 3.9 to 5.5 mmol / L (70 to 100 mg / dl).

Chemistry of carbohydrates

The term carbohydrate refers to hydrates of carbon and is derived from the observation that the empirical formulas for these compounds contain approximately one molecule of water per carbon atom. In more descriptive terminology, the carbohydrates are defined as the aldehyde and ketone derivate of polyhydric alcohols. The simplest carbohydrate is glycoaldehyde, the aldehyde derivatives of ethylene glycol. The aldehyde and ketone derivates of glycerol are glyceraldehyde (glycerose) and dihydroxyacetone, respectively.

The formula for glucose may either be written in aldehyde or enol form. The presence of double bond and a negative charge in the enol anion form make glucose an active reducing substance and provide a basis for its analytical determination. Thus, glucose in hot alkaline solution readily reduces metallic ions such as cupric ions and the color change can be used as a presumptive indication for the presence of glucose. Sugars capable of reducing cupric ions in alkaline solution are commonly known as reducing sugars.

Monosaccharides – are sugars containing, 3,4,5 and 6 or more carbon atoms. Aldehyde derivative are called aldoses and ketone derivatives are called ketoses.

Typical examples are the six–carbon sugars, glucose (an aldose) and fructose (a ketose).

Disaccharides are sugars formed by interaction of groups between two monosaccharides with loss of a molecule of water. The chemical bond between saccharides always involves the aldehydes or ketone group of one monosaccharides joined to the other monosaccharides, either by latter’s aldehyde or ketone group (e.g. sucrose) or by latter’s alcohol group (e.g. maltose). The linkage of an aldehyde or ketone with alcohol is called glycosidic linkage.

                Example of common dissacharides:

1.       Maltose (glucose and glucose)
2.       Lactose (glucose and galactose)
3.       Sucrose (glucose and fructose)

Polysaccharides result from the linkage of many monosaccharide units together.

Metabolism of carbohydrates

Starch and glycogen are partially digested by the action of salivary amylase to form intermediate dextrin and maltose. Amylase activity is inhibited at the acid pH of the stomach. In the intestine, the pH is increased by alkaline pancreatic juice and the amylase of the pancreas affects digestion of starch and glycogen to maltose. The latter, along with any ingested lactose and sucrose, is split by the dissacharides in the intestinal mucosa (maltase, lactase and sucrose) to form the monosaccharide glucose, galactose and fructose.

Following absorption into the portal vein, the hexoses are transported to the liver. Depending on the needs of the body, the carbohydrates may be converted to keto acids, amino acids and protein or converted to fat and stored as adipose tissue.

Terms used to describe general processes in carbohydrate metabolism:

1.       Glycogenesis – conversion of glucose and other hexoses to glycogen by hepatic cells.
2.       Glycogenolysis – breakdown of glycogen to form glucose and other immediate products.
3.       Gluconeogenesis – formation of glucose from non – carbohydrate sources such as amino acids, glycerol or fatty acids.
4.       Glycolysis – conversion of glucose or other hexoses into lactate or pyruvate. S

Functions of carbohydrate:

1.       Providing chemical energy to the body.
2.       Furnishing part of the structural integrity of the cell.
3.       Determining blood types.

Stability of glucose in body fluids:

1.       Glucose is unstable when blood is permitted to clot and stand uncentrifuged at room temperature and the average decrease is about 7% per hour.

2.       In separated, unhemolyzed serum, the glucose concentration is stable up to 8 hours at 25oC and up to 72 hours at 4oC.

3.       The addition of sodium fluoride or iodoacetate will prevent glycolysis by inhibiting phosphogyceraldehyde dehydrogenase and allow the glucose to be stable for 24 hours at room temperature.

4.       Fluoride ions inhibit urease activity and should therefore not be used for urea determinations that require urease.

5.       Glucose level is arterial blood is higher than in venous blood, while the capillary blood approximates arterial blood. Glucose value in serum or plasma is about 10 – 15% higher than in whole blood.

6.       Plasma of the whole blood fraction is the choice in glucose determination.

7.       Cerebrospinal fluid glucose is about 2/3 of that of whole blood sugar.

Regulation of blood glucose concentration

In the fasting state, the level of blood glucose is maintained by drawing upon the glycogen stores of the liver and a slight amount may also be derived from the kidneys. Both of these organs contain the specific enzyme, glucose–6–phosphatase necessary for conversion of glucose–6–phosphate to glucose.

Skeletal muscles, although it stores glycogen, is lacking in this enzyme and cannot directly contribute to glucose in blood.

As blood glycose level increases, usually by absorption of carbohydrates from intestines, glycogenolyis is replaced by glycogenesis, whereby excess blood glucose is converted into liver and muscle glycogen.

Hormones that regulate blood glucose concentration


Insulin

Produced by the beta cells of the pancreatic islets of Langerhans in response to an elevated blood glucose level. It is the only hormone that lowers blood glucose. It also alters the metabolic pathways of glucose metabolism by enhancing the formation of glycogen, fat and proteins.

Glucagon

Produced by the alpha cells of the pancreatic islet of Langerhans in response to a low blood glucose level. It is the principal hormone for producing a rapid increase in the concentration of glucose in the blood. It does so by stimulating hepatic glycogenlysis and gluconeogenesis but has no effect on muscle glycogen.

Somatostatin

Produced by the delta cells of the pancreatic islet of Langerhans and inhibits secretion of insulin and glucagon, thereby modulating their reciprocating action. Somatostatin only has minor effect on the blood glucose concentration.

ACTH

Adrenocorticotropic hormone (ACTH), also called corticotropin, is a small polypeptide secreted by the anterior pituitary. Like GH, it increases the concentration of blood glucose because of its antagonistic action towards insulin.

Growth Hormone

Also called somtotropin and is produced by the anterior pituitary. The action of GH is antagonistic to that of insulin in that it inhibits glucose uptake by the tissues and stimulates liver glycogenlysis, thus raising the blood glucose concentration.

Cortisol and 11–deoxysteriods

Secreted by adrenal cortex and raises blood glucose concentration primarily by stimulating glyconeogenesis. They also have some metabolic effects that are antagonistic to insulin and are sometimes referred to as diabetogenic hormone.

Epinephrine

Also known as adrenaline, is a catecholamine secreted by adrenal medulla. It increases the blood glucose level by stimulating glycogenolysis and serves as a back up for glucagon. It is triggered by physical or emotional stress. This causes immediate increase in the production of glucose for energy along with an increase in heart rate, blood pressure and other physiologic effect.

Thyroxine (T4)

A tetraiodinated amino acid secreted by the thyroid gland. It promotes glycogenolysis and can lead to a depletion of glycogen store in the liver. It also accelerates glucose absorption from the intestine and may lead to a mildly abnormal, diabetic type of glucose intolerance in hyperthyroid individuals even though their fasting blood glucose level is usually normal. Although the action of thyroxine is hyperglycemic, it has an insignificant role in regulating the blood glucose concentration.

Somatomedins

These are peptides in the liver in response to stimulation of growth hormone. Somatomedins are a group of hormones, including somatomedin A, somatomedin C and insulin like growth factors I and II, that directly promote growth. In addition to their growth–promoting effects, somatomedins show insulin–like activity in some tissues, such as adipose tissue. It has been shown that insulin – like growth factor I and II has a structure similar to that of insulin.

******  Methods of glucose determination  ******

1.     Chemical Methods

a.      Alkaine Copper Reduction Methods

In hot alkaline solution, cupric ions are reduced to cuprous ions by glucose with the formation of cuprous oxide. Other reducing sugars are lactose, fructose and pentose. Sucrose is a non–reducing sugar.

(1)  Folin–Wu

The tungstic acid filtrate of whole blood / plasma / serum is heated with alkaline copper solution. The glucose in the solution reduces cupric ions and is made to react with phosphomolybdic acid to form a blue complex of molybdenum blue which is measured colorimetrically and compared with a standard.

This method lacks specificity since it also measures saccharoids like ergothione, glutathione, ascorbic acid, uric acid and creatinine.

A constricted tube is used in this test to minimize surface tension and thus prevent the reoxidation of cuprous ions by oxygen. Eighteen percent sodium sulfate may also be added to decrease the solubility of oxygen and thus achieve the same effect as the constricted tube.

Normal values:        80 – 120 mg/dl

(2)  Nelson–Somogyi

This is the most accurate redox method and believes to be a measure of true glucose, because of the saccharoid free PFF. The barium sulfate formed acts as an adsorbent to which saccharoids adhere. In this method, the PFF is made to react with an arsenomolybdate reagent forming a blue end product of arsenomolybdate blue.

Normal values:        65 – 100 mg / dl

(3)  Neocuproine

PFF is made to react with dimethyl phenanthroline hydrochloride or neocupreine forming a yellow to yellow–orange cuprous–neocupreine complex.

(4)  Benedict’s Method

Same as Folin–Wu except that sodium sulfite is added which increases the sensitivity of glucose at the expense of saccharoids and uses a copper reagent of Benedict. The copper reagent is a modification of the Fehling in which the alkalinity is diminished by using sodium carbonate instead of the hydroxide and the addition of alanine. The amino acid forms a cupric salt complex which is unaffected by the non–glucose substances of the blood.

(5)  Shaeffer–Hartman–Somogyi (Iodometric method)

Cuprous ions formed react with iodine in acidic solution and excess iodine in the blank and sample is titrated with thiosulfate. The difference is equal to the reducing sugar present in the sample.

Advantages:

(a)   A colorimeter is not required.
(b)  It is considered to be the most accurate for the determination of glucose.

Disadvantage:

(a)   It is time consuming.

b.     Alkaline Ferric Reduction Methods

(1)  Hagedorn–Jensen (Autoanalyzer method)

In a hot alkaline solution, yellow ferricyanide ion oxidize glucose to a colorless ferrocyanide ion. Also known as inverse colorimetry.

Dialysis separates the glucose from red blood cells and proteins. The dialyzed glucose decolorizes the potassium ferricyanide to ferrous from and the disappearance of color which is proportional to the amount of glucose of color which is proportional to the amount of glucose is measured photometrically.
   


c.      Condensation Methods

(1)  Ortho–toluidine method (Dubowski)

o–toluidine condenses with the aldehyde group of glucose in a hot acetic acid solution to form an equilibrium mixture of a glycosylamine and the corresponding Schiff base forming a green chromogen measurable at 630nm.

This is the most specific non–enzymatic method for glucose determination. The hot acidic solution is for enolization or enol formation. However, its use presents a health hazard because o–toluidine is now classified as carcinogen.

Sources of error:

(a)   Bilirubin gives falsely elevated values since it may be partially converted to green pigment biliverdin

(b)  Turbidity in the final solution owing to presence of lipemia or the plasma expander dextran in the specimen likewise causes falsely high results.

(c)   Sodium fluoride and EDTA also contribute to the final color of the reaction

(d)  Somewhat higher values are obtained in patients with uremia and galactosemia.

(2)  Phenol method


                                                                                                      
2.     Enzymatic Method

a.      Glucose oxidase method

(1)  Saifer–Gerstenfield method

Glucose is measured by the reaction with glucose oxidase in which gluconic acid and hydrogen peroxide are formed. Hydrogen peroxide then reacts with an oxygen receptor such as ortho–toluidine or ortho–dianisidine in a reation catalyzed by peroxidase to form a blue color.




(2)  Trinder method

Utilizes a dye, 4–aminophenazone oxidatively coupled with phenol but has the same principle as Saifer–Gerstenfield.

(3)  Gochman method

Utilizes a dye, 3–methyl–2–benzolinome hydrazone (MBTH) oxidatively coupled with N,N–dimethylaniline (DMA) but has the same principle as Saifer–Gerstenfield.

(4)  Polarographic method

The amount of oxygen consumed in the oxidation of glucose to gluconic acid is measured using a polarographic oxygen electrode. This method is precise, linear and free from interferences. Whole blood should not be used since viable cells use oxygen.

(5)  Dextostix

These are reagent strips made of firm cellulose strips impregnated with highly purified glucose oxidase and chromogen indicator systems under a semi–permeable membrane which permit approximate quantitation of blood glucose.

b.     Hexokinase method

This is the most specific method for blood glucose and is the reference method. This system involves two coupled reactions:

         
                     
(1)  NADP+ is required as the cofactor when glucose–6–PO4 dehydrogenase is derived from yeast.

(2)  NAD+ is used instead of NADP+ when the source of glucose–6–PO4 dehydrogenase is bacterial (Leuconostoc mesenteroides)

(3)  Hemolysis interferes with hexokinase system because red blood cell glucose–6–PO4 dehydrogenase and 6–phosphogluconate dehydrogenase use NADP+ as substrate.

(4)  The hexokinase system can also be coupled to an indicator reaction using phenazine methosulfate (PMS) and an iodonitrotetrazolium (INT) so that absorbance can be measured in the visible range.

c.      Sunderman catalase method

Makes use of catalase, the evolved H2O2 in the presence of catalase oxidizes methanol to formaldehyde with the latter measured by a chromatographic acid giving a blue violet color complex.

******  Tests for Diabetes Mellitus  ******

1.      Glucose Tolerance Test – a multiple blood and urine sugar test that rules out diabetes. This is to detect the presence of diabetes in a patient who is suspected to be diabetic but whose ordinary routine FBS determination is not or is slightly elevated.

Patients with mild or diet–controlled diabetes may have a fasting blood glucose levels within normal range but unable to produce sufficient insulin for prompt metabolism of ingested carbohydrates. As a result, blood glucose roses to abnormally high levels and the return to normal is delayed. In other words, the patient has decreased tolerance for glucose. Therefore, glucose tolerance test are most helpful in establishing a diagnosis of mild case of diabetes.

Principle:            A normal individual when given a glucose challenge is capable of converting  
                                the same to glycogen and his glucose level is therefore back to normal in 3
                                hours. A diabetic person (because of insulin deficiency) will remove glucose
                                from the breakdown at a rate slower than that of a normal individual.

a.      Oral Glucose Tolerance Test (OGTT)

(1)  Janney Isaacson Method or Single Oral Dose Method

(2)  Exton–Rose Method or Divided Oral Dose Method

Procedure for OGTT:

(1)  The patient should have unlimited physical activity and an unrestricted diet containing at least 150 grams of carbohydrates for 3 days before the test is performed.

(2)  The test should be performed in the morning after the patient has fasted for 10 – 16 hours.

(3)  A fasting blood sample and urine specimen is obtained. This will serve as baseline.

(4)  A solution containing 0.5 grams of glucose per pound body weight is given to children and a solution containing 100 grams of glucose is given to adults. One hundred grams of glucose is dissolved in about 200 ml of water and flavored with lemon juice. Commercially prepared orange juices are now available for this kind of procedure.

(5)  Blood and urine specimens are collected at 30 minutes, 1 hour, 2 hours and 3 hours for analysis for glucose. Normally, all these urine specimen show a negative reaction. The level of the plasma glucose at which glucose appears in the urine is called the renal threshold and is approximately 130 mg / dl.

b.     Intravenous Glucose Tolerance Test

Since glucose absorption after the oral ingestion take place through the small intestine, it follows that, when gastric or intestinal disease is present, the rate of absorption will be affected. This is the basis for the intravenous method. The same precautions and procedures as the oral method are followed except for the administration of glucose which is introduced into the vein.

                Normal response to Glucose Tolerance Test


                                                                                                Blood Sugar                                    Urine
                                                                                                (mg/dl)

                Fasting                                                                 80                                                           0

                After 30 minutes                                             155                                                        0

                After one hour                                                  145                                                        0
               
                After two hours                                               75                                                           0
               
                After 3 hours                                                     80                                                           0

                In normal persons:

a.       The fasting blood specimen is normal.
b.       The 30 minute blood specimen does not exceed the fasting blood specimen by more than 75 mg.
c.       The one hour blood specimen does not exceed the 30 minutes blood specimen by 10 mg.

·         In diabetes, the one hour blood specimen exceeds the two hours specimen by 10 mg or more and the urine specimens are usually positive for glucose.

2.     Post–prandial Blood Glucose Test

It is based on the principle that the glucose concentration in blood specimens drawn 2 hours after a meal is rarely elevated in normal individuals, while it is significantly increased in diabetic patients.

3.     Insulin Tolerance Test

Insulin administered to a normal person in the post–absorptive state causes a prompt decrease of blood sugar and then a gradual return to the original level. Normally, the blood sugar level falls to about 50% of the fasting level in 30 minutes and return to the original level or above in 2 hours.

4.     Epinephrine Tolerance Test

Epinephrine accelerates glycogenolysis and promptly increases the blood sugar. This increase of blood sugar following administration of epinephrine is an index of the quantity and availability of liver glycogen for maintaining normal blood sugar. Ten minims of 1:1000 solution of epinephrine hydrochloride is injected intramuscularly after a fasting blood specimen has been taken.

5.     Tolbutamide Diagnostic Test

This test is based on the difference in response to normal subjects and diabetic individuals to intravenous administration of a test dose to tolbutamide. Tolbutamide is a compound that stimulates the pancreas to produce insulin. This test differentiates insulinomas from hyperinsulinemia states.

Twenty ml of orinase solution (Tolbutamide) at a constant rate over a 2–3 minute period is injected intravenously after a fasting blood specimen has been taken. The blood specimen are taken at exactly 20 and 30 minutes later, timing from the mid–point of the injection and the blood sugar is taken after feeding a high carbohydrate breakfast.

Interpretation:

If the blood glucose value of the 20 minute specimen is 90% or more of the fasting level, the patient is definitely diabetic. If the 20 minutes specimen is within the range of 85–89% of the fasting level, diabetes is probable; the range of 75–84% represents borderline cases and less than 75% represents normal. The 30 minutes specimen is of value in confirming the diagnosis.

  CLINICAL SIGNIFICANCE OF CARBOHYDRATE DETERMINATION


I.  Hypoglycemia

Hypoglycemia is a syndrome characterized by low plasma glucose levels, usually less than 50 mg/dl (2.8 mmol/L), although not all investigator agree on the exact cutoff values.

Classification of hypoglycemia

A.     Reactive hypoglycemia – occurs because of some stimulus and is caused by:

1.      Factitious hypoglycemia – excessive administration of insulin or other hypoglycemic agents or by a reduction in gluconeogenesis as a result of ethanol ingestion.

2.      Postprandial hypoglycemia – occur several hours after a meal in individuals who have had gastrointestinal surgery or have mild diabetes. Relief is obtained by food intake.

B.     Fasting or Spontaneous hypoglycemia – caused by excessive insulin secreted by
insulin –producing pancreatic islet cell tumors (insulinomas), non–pancreatic tumors that produce substances with insulin–like activity, hepatic dysfunction, glucocorticoid deficiency, sepsis or depleted glycogen stores.

Symptoms of hypoglycemia

A.     Rapid fall of plasma glucose (adrenergic symptoms)

1.       Sweating
2.       Weakness
3.       Shakiness
4.       Trembling
5.       Nausea
6.       Hunger
7.       Rapid pulse
8.       Light–headedness
9.       Epigastric discomfort

B.     Gradual fall of plasma glucose to less than 20 or 30 mg/dl (neuroglycopenia)

1.       Headache
2.       Confusion
3.       Lethargy
4.       Seizure
5.       Unconsciousness
6.       Irreversible brain damage

Causes of hypoglycemia in neonates and children

A.      Eclampsia
B.      Prematurity
C.       Polycythemia
D.      Respiratory distress syndrome
E.      Gluconeogenic enzyme or counter regulatory hormone deficiencies
F.       Galactosemia
G.      Hereditary fructose Intolerance

II.  Hyperglycemia

Diabetes mellitus is the most important disease associated with hyperglycemia. It is characterized by a deficiency of insulin secretion or action.

Classification of Diabetes mellitus & other glucose intolerance:

               
Primary Diabetes mellitus – based on the activity and state of the pancreas involving the insulin–producing cells.

A.     Insulin Dependent DM (IDDM) or Type I DM:

1.       Occurs at an early age (juvenile onset).
2.       Seen in thin patients.
3.       Abrupt onset of symptoms.
4.       Absolute deficiency of insulin.
5.       Congenital – not hereditary
6.       Ketosis prone.
7.       Requires insulin treatment.

B.     Non–insulin dependent  (NIDDM) or Type II DM:

1.       Occurs after age 40 (maturity onset)
2.       Seen in obese patients
3.       Gradual onset of symptoms
4.       A degree of basal insulin production persists.
5.       Hereditary – genetically linked.
6.       Ketosis resistant
7.       Requires drugs that potentiate insulin release like oral hypoglycemic agents.

Signs and symptoms of Diabetes mellitus: The classical triad

1.       Polyuria – excessive urination

Ketosis – overproduction of ketone bodies and result in appearance either in urine (ketonuria) or blood (ketonemia). Since acetone is volatile, it may be present in the breath of diabetics, giving it a characteristic sweet “organic” odor.

2.       Polydipsia – intake of large volume of water.

3.       Polyphagia – excessive desire to eat

Post–treatment complications of Diabetes mellitus:

1.       Retinopathy leading to blindness, kidney failure, neurologic defects and microvascular and macrovascular disease.

2.       Heart attacks and strokes due vascular complications

3.       Coronary artery disease

4.       Gangrene due to diminished blood flow to the legs and feet because of arteriosclerosis causing lower limb amputations along with loss of response to normal pressure, minor trauma, susceptibility to infections, etc.

Secondary Diabetes mellitus – associated with endocrinopathies.

A.      Cushing’s syndrome – a disease of the adrenal cortex causing excessive secretion of diabetogenic glucocorticoid.

B.      Pheochromocytoma – a tumor involving the chromocytes of the adrenal medulla causing excessive production of epinephrine and norepinephrine.

C.       Acromegaly – a disease characterized by enlargement of the bones of the hands, feet and skull due to excessive amount of growth hormone.

Impaired Glucose Tolerance (IGT) – is characterized by glucose levels that are not normal yet sufficiently abnormal to be classified in the category of diabetes mellitus. The glucose levels of these patients may revert to normal or remain borderline, but those patients do have a greater risk for the development of diabetes mellitus.

Gestational diabetes mellitus (GDM) – is characterized by the onset of or IGT during pregnancy. After delivery, the patient’s glucose level may revert to normal or the patient may gent diabetes mellitus later in life.

Previous abnormality of glucose tolerance (PrevAGT) – is used to refer to individuals who now have a normal glucose levels but who have previously had abnormal glucose tolerance.

e.g.         Women who have GDM
                                  Obese individuals with NIDDM

Potential abnormality of glucose tolerance (PotAGT) – pertains to persons who have normal glucose levels but who are at increased risk for the development of diabetes mellitus.

e.g.         Identical twins
                                  Sibling or offspring of diabetic patient

III.  Inborn errors of carbohydrate metabolism

A.    Glycogen storage disease



B.     Disorder of fructose metabolism

1.       Hereditary fructose intolerance – caused by a deficiency of fructose–1–phosphase aldolase, thus causing the accumulation of fructose–1–phosphate in cells. The ingestion of fruit or sucrose produces vomiting, hypoglycemia, hepatomegaly and failure to thrive.

2.       Fructose–1,6 diphosphate deficiency – results from lack of fructose 1,6– diphosphatase, an enzyme necessary in the formation of glucose from pyruvate. Infants with this rare disease have fasting hypoglycemia, lactic acidosis, hepatomegaly and a poor prognosis.

3.       Essential fructosuria – benign condition caused by a deficiency of hepatic fructokinase. It is characterized by high fructose levels in serum and urine after ingestion of sucrose or fructose.

C.     Mucopolysaccharide storage disease

Mucopolysaccharides are structural components of cartilage, bone, skin and other connective tissues. They consist of repeating disaccharide units that contain a hexosamine (usually acetylated), a uronic acid and often a sulfate group attached to the hexosamine. The three classes of mucopolysaccharides are dermatan sulfate, heparan sulfate and keratan sulfate.

Mucopolysaccharides are hereditary disorders caused by a deficiency of one or more lysosomal enzymes. The mucopolysaccharides (glucosaminoglycans) accumulate in various tissues and are excreted in the urine.

Hurler’s syndrome is the prototype of all mucopolysaccharide storage diseases. It is a severe, progressive disorder characterized by corneal clouding and death usually before the age of 10. Individuals with this disorder have coarse faces, skeletal abnormalities, developmental delay and hepatosplenomegaly.

IV.  Glycated hemoglobin

Glycated hemoglobin provides an index of the mean concentration of blood glucose over the preceding two months. The test is useful in determining compliance with therapy and the extent to which satisfactory diabetic control has been achieved.

Formation of glycated hemoglobin

HbA contains several minor hemoglobin components, identified as HbA1a, HbA1b and HbA1c. These are modifications of HbA and are collectively referred to as glycated hemoglobin, glycosylated hemoglobin, “fast hemoglobin” or glycohemoglobin.

HbA1c is the most defined by the hemoglobin fraction and is formed by a non–enzymatic reaction, referred to as glycation, between glucose and the N–terminal valine amino acid of each beta chain of HbA to form a labile Schiff’s base (pre–A1c) with an aldininie structure. As the red cell circulates, some of the aldiminine undergoes a slow, irreversible Amadoric arrangement to yield a stable ketoamine (HbA1c).  This reaction is continuous over the 120–day life span of the RBC and is proportional to the concentration of glucose in blood.

Methods of determining glycated hemoglobin

1.       Methods based on charge differences

a.       Ion exchange chromatography
b.       High performance liquid chromatography
c.       Electrophoresis
d.       Isoelectric focusing

2.       Methods based on chemical reactivity

a.       Hydroxymethylfurfural (HMF) / thiobarbituric acid colorimetric method

3.       Method based on structural differences

a.       Affinity chromatography

Normal values:                   HbA1                     5 – 8%
                                                HbA1c                    3 – 6%



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