GLYCOGEN STORAGE DISEASE

Glycogen storage diseases are the result of deficiency of enzymes that cause the alteration of glycogen metabolism. The liver forms (type I, III, IV and VI) are marked by hepatomegaly due to increased liver glycogen and hypoglycemia caused by inability to convert glycogen to glucose. The muscle forms (type II, IIIA, V and VII) have mild symptoms appearing during sternous exercise owing to inability to provide energy for muscle contraction.

INBORN ERROR OF CARBOHYDRATE METABOLISM

Deficiency or absence of an enzyme that participate in carbohydrate metabolism may result in accumulation of monosaccharides, which can be measured in urine. Most of these conditions are inherited as autosomal recessive traits.

DISORDER OF GALACATOSE METABOLISM
Galactose is derived from milk in diet. It is the C4 epimer of glucose. A deficiency of any of the enzyme that participates in conversion of galactose to glucose results in galactosemia. Galactosemia occurs due to inhibition of glycogenolysis.

GALACTOSE-1-PHOSPHATE URIDYL TRANSFERASE DEFICIENCY

Infants with this deficiency fail to thrive on milk because half of the milk sugar, lactose is galactose. Within few days of milk ingestion neonates manifest vomiting and diarrhea. Failure to thrive, liver disease, cataracts and mental retardation develop later. This disorder is identified by measuring erythrocyte galactose -1-phosphate uridyltransferase activity.

GALACTOKINASE DEFICIENCY

This is milder condition manifested by cataracts caused by galactitol deposits in the lens. The diagnosis is confirmed by demonstrating normal transferase activity no galactokinase in red blood cells.

DISORDER OF FRUCTOSE METABOLISM
Fructose may appear in the urine after eating fruits, honey, and syrups, but has no significance in these conditions. Three disorders of fructose metabolism inherited as autosomal recessive trait produces fructosuria.

Essential fructosuria
This occurs due to deficiency of fructokinase

Hereditary fructose intolerance

A deficiency of fructose-1-phosphate aldolase produces this disorder with hypoglycemia and liver failure. Fructose ingestion inhibits glycogenolysis and gluconeogenesis, producing hypoglycemia.

Hereditary fructose-1, 6-diphosphate deficiency

DISORDER OF PENTOSE METABOLISM

Alimentary pentosuria
Pentose may be present in the urine after eating large quantities of fruits such as cherries, plums, or prunes.

Essential pentosuria
This is harmless inborn error caused by deficiency of L-xylulose reductase an enzyme involved in the glucuronic acid pathway.
Individual sugars can be measured by qualitative tests and chromatography

Emergency treatment of hypoglycemia

Glucose should be administered orally (10-20 g in adult patient, 3 times before giving a meal). If oral therapy is not possible the parenteral dose of glucose for adult should be 25-50g as 50-100 mL of 50% dextrose should be given. Failure to respond to glucose, glucagon should be administered intramuscularly or intravenously, steroids (hydrocortisone).

Causes of hypoglycemia:

1.    Medical therapy of diabetes especially insulin administration or oral hypoglycemia drug is the most common cause of fasting hypoglycaemia.

2.   Surreptitious (self-induced) administration of hypoglycemic agents (factitious or felonious hypoglycaemia) like insulin, sulphonylureas, metiglinides, etc.

3.      Insulinoma: Insulin producing islet cell tumors.

4.      Autoimmune hypoglycemia 

In one condition antibodies binds to insulin receptors and mimic the action of insulin. Laboratory finding shows high plasma insulin concentrations but suppressed C-peptide and proinsulin. The other syndrome, autoimmune insulin syndrome, in which antibodies are direction towards insulin. Laboratory finding shows high plasma concentration of insulin and C-peptide (C-peptide level is quiet less than insulin).

1.      Hypoglycaemia associated with renal failure:

Renal impairment leading to hypoglycaemia is the second most common cause of hypoglycaemia, after insulin therapy. The most important factor here is calorie restriction. In normal subjects, the kidney, by gluconeogenesis supply 45% of glucose during prolonged starvation. In uraemic patient this process is impaired. Other mechanisms include increase insulin half-life due to impaired renal clearance and degradation.

2.      Hypoglycemia associated with liver disease:

Liver can maintain glucose homeostasis even functioning liver mass reduces to <20% and hypoglycemia does not occur unless liver is extensively damaged. Conditions like fatty liver, cirrhosis, infective hepatitis, hepatocellular carcinoma are associated with hypoglycaemia.

3.      Alcohol induced hypoglycaemia: 

Alcohol induced fasting hypoglycemia is due to direction inhibition of gluconeogenesis. This is due to accumulation of NADH and increased NADH/NAD⁺ ratio resulting from the oxidation of ethanol. Alcohol induced fasting hypoglycemia usually develops 6-36h after ingestion of alcohol. There is severe metabolic acidosis with high blood lactate. Hyperketonaemia and ketonuria are present predominantly β-OHB, since the accumulation of NADH suppress the conversion of it to acetoacetate. Prompt IV glucose treatment should be done.

Alcohol potentiates the hypoglycemic effect of insulin and sulphonylurea drugs. Alcohol potentiates the insulin-stimulating effect of glucose and thus increase the risk of reactive hypoglycemia. This is seen during consumption of alcohol and sucrose (e.g. in syrup or tonic) in empty stomach and followed by not eating for few hours afterward. This effect is not seen when saccharin or fructose is substituted for sucrose as sweetening agent. Starchy foods like breads increase the risk fro reactive hypoglycaemia, whereas foods providing fat or protein have the reverse effect.

During exercise, during the first 5-10 minutes of severe exercise, muscle glycogen is the source of energy, by 40 min, 75-90% of glucose is supplied by blood, mainly from increased hepatic glucose production (75% from glycogenolysis and 25% from gluconeogenesis).

4.      Reactive (alimentary) or postprandial hypoglycemia

This occur after gastric surgery, antibodies to insulin, inborn error of metabolism. Symptoms occurring 2-4h after food ingestion and last for about 10-20 min. This is also seen in patients with hereditary fructose intolerance after ingestion of fructose.

Hypoglycemia in Diabetes Mellitus

Hypoglycemia occur frequently in both type 1 and 2 diabetes. This occurs in diabetic patients using hypoglycemia drugs or insulin. In many patients with type 1 disease do not experience the neurogenic warning symptoms for years and are prone to severe hypoglycemia this is called hypoglycemia unawareness. 

HOW TO IDENTIFY THE CAUSE OF HYPOGLYCEMIA

IDENTIFICATION OF CAUSE OF HYPOGLYCEMIA

PLASMA INSULIN AND C-PEPTIDE

Increase in Insulin and C-peptide in the presence of hypoglycemia indicates islet-cell tumors, autoimmune insulin secretion, and drug-induced (sulphonylureas, repaglinide) causing endogenous hyperinsulinaemia.

Decrease in insulin and C-peptide indicates presence of other secondary conditions like chronic renal failure (as C-peptide is excreted by kidney), liver disease, alcohol induced, anorexia nervosa, etc.

Increase in insulin but decrease in C-peptide indicates administration of exogenous insulin, Insulin anti-receptor antibodies (IR-A).

PLASMA Β-HYDROXYBUTYRATE

Hypoglycemia due to hyerinsulinemia shows low ketone bodies. In hypoglycemia due to other conditions like liver disease, anorexia nervosa, hypopituitarism etc, this ketone body is raised.

PLASMA PROINSULIN

Normally only <20% of insulin is released in circulation. In islet cell tumor, circulating proinsulin is increased.

INSULIN ANTIBODIES

The presence of insulin antibodies, due to pre-exposure to exogenous insulin may give false high plasma insulin concentrations. Since C-peptide does not cross-react with insulin antibodies, its measurement can be used as index of β-cell function. 

How Hypoglycemia is investigated ?

A venous plasma glucose concentration below 50 mg/dl is called hypoglycaemia. The diagnosis of hypoglycemia necessitates the presence of Whipple’s triad. This consists of:

Fig. Whipple's Triad

1)   Symptoms of hypoglycemia

2)   Low plasma glucose concentration and

3)   Symptoms relieved by glucose administration.

Fig. Classical Signs and symptoms of Hypoglycemia

The classic signs and symptoms of hypoglycemia are trembling, sweating, nausea, rapid pulse, lightheadedness, hunger and epigastric discomfort. Neuroglycopenia can be seen in severe cases (headache, confusion, blurred vision, dizziness, and seizures).

The most common cause of hypoglycemia are drugs like propranolol, salicylate, oral hypoglycemic drugs with long half life like chlorpropamide, insulin secreting sulfonylureas, glycogen storage disease, alcoholism, septicemia, hepatic failure, Addison’s disease etc. 

REGULATORY RESPONSE TO HYPOGLYCEMIA

In hypoglycemia, the shortage of glucose in neurons activates hypothalamus, and an autonomic response to restore and maintain glucose supply initiates which has many effects like:

ACTIVATION OF SYMPATHETIC NERVOUS SYSTEM

 α-ADRENERGIC EFFECTS

• Inhibition of endogenous insulin release
• Increased cerebral blood flow (peripheral vasoconstriction)

 β-ADRENERGIC EFFECTS:

• Stimulation of glycogenolysis
• Stimulation of glucagon release (also α cells can sense directly)
• Stimulation of lipolysis
• Inhibition of muscle glucose uptake
• Increased cerebral blood flow (by increasing cardiac output)

CATECHOLAMINE RELEASE FROM ADRENAL MEDULLA

• Potentiates the α and β adrenergic effects

ACTIVATION OF PARASYMPATHETIC NERVOUS SYSTEM

• Stimulates vagus nerve
• Stimulation of gastric acid secretion
• Stimulation of parotid salivary secretion. 

There is hierarchy of response of counter-regulatory hormones; glucagon, epinephrine, cortisol and GH. Glucagon and epinephrine are rapidly acting hormones whereas latter two are slow acting and are active at late phase of hypoglycemia. During fast state the first mechanism is inhibition of endogenous insulin secretion and followed by release of counter regulatory hormones in hierarchy.

Decreased endogenous insulin occurs at glucose level 80 mg/dl; increase glucagon, adrenaline, cortisol and GH secretion at 60 mg/dl and development of hypoglycaemic symptoms occurs at 50 mg/dl and impairment of cognitive function at 40 mg/dl. 

INVESTIGATION OF HYPOGLYCEMIA

First is demonstration of hypoglycemia and second to identify the cause of hypoglycemia.

DEMONSTRAITON OF HYPOGLYCEMIA

MEASUREMENT OF BLOOD GLUCOSE

Measurement of blood glucose (insulin, C-peptide) during acute neuroglycopenia (characterized by sweating, anxiety, hunger, palpitation and weakness) is the best test for the diagnosis of hypoglycemia.

PROVOCATION TEST:

Prolonged fast:

This is the single most useful test to evaluate suspected hypoglycemia. The aim of this test is to demonstrate spontaneous hypoglycemia in the presence of neuroglycopenic symptoms during prolonged fasting for 48 h, and that the symptoms resolve on glucose administration.

During the fasting period blood glucose, insulin, C-peptide is measured at every 4-6 hours. But as glucose level falls below 50 mg/dl frequent sample must be taken. About 95% of patient will develop hypoglycaemia within 48 h. Measurement of β-hydroxybutyrate and its raising presence indicates suppression of insulin release and fast can be terminated by giving glucose when FBS becomes <45 mg/dL and patient exhibit signs or symptoms of hypoglycemia.

Mixed meal test:

This is used to investigate patients who experience postprandial symptoms, for the possibility of reactive hypoglycemia. Meal is ingested and plasma glucose measured every 30 min for 6h and at any time during symptomatic phase. Patients developing neuroglycopenia symptoms during hypoglycemia, but not at other times during the test, are considered to have postprandial hypoglycemia. 

COMPLICATIONS OF DIABETES

DIABETIC RENAL DISEASE (DIABETIC NEPHROPATHY)

It is most common in type 1 diabetes. Some 20-30% of patients with type 1 diabetes will develop renal disease (15-25 years after diagnosis). It is less prevalent in type 2 diabetes (only 10-20% lifetime risk).

HYPERFILTRATION AND MICROALBUMINURIA

The earlier symptoms of diabetes includes hyperfiltration (with urine albumin excretion, UAE, <30mg/24 hour or 20µg/min) followed by progression through microalbuminuria to proteinuria (UAE>300 mg/24 h or 200µg/min). After this GFR falls and progress to ESRF. The first and best opportunity to detect the disease clinically is at the stage of microalbuminuria. Dip-stick testing or urine is not usually positive at such concentration of albumin and detection relies on either 24 h quantitation or more conveniently the use of albumin/creatinine ratio (normal <2.5 mg/mmol in men and <3.5 mg/mmol in women) on at least two out of 3 separate urine specimens over a 3-6 month period can be done. Due to day to day variation of UAE rates 2 of 3 samples should be positive for the diagnosis. Microalbuminuria is not just a risk factor of nephropathy but an independent risk factor for CAD (one of the most potent risk factors known), being also associated with dyslipidaemia, hypertension, endothelial dysfunction and diabetic retinopathy.

TYPE 4 RENAL TUBULAR ACIDOSIS

Hyporeninaemic hypoaldosteronism may be a manifestation of diabetic nephropathy. It presents with hyperchloraemic, hyperkalaemic metabolic acidosis. Failure of renin to rise in response to posture or sodium restriction suggest an interstitial (juxtaglomerular) defect. The failure of aldosterone release to be stimulated directly by resulting hyperkalaemia suggest the possibility of dysfunction of adrenal zona glomerulosa.

CHARCOT FOOT

It is a specific foot deformity occurring due to neuropathy and if untreated leads to bone collapse of the foot causing outward bowing. 

OTHER DIABETIC EMERGENCIES: HYPEROSMOLAR HYPERGLYCAEMIC STATES AND ALCOHOLIC KETOACIDOSIS

HYPEROSMOLAR HYPERGLYCAEMIC STATES

Initially called hyperosmolar non-ketotic (HONK) hyperglycaemia. The dominant clinical feature is dehydration. It mainly occurs in older subjects with type 2 diabetes mellitus. The cycle of hyperglycaemia, dehydration (occurring due to vomiting, polyuria, glycosuria osmotically takes more water in urine) and increased counter regulatory hormones (induced by acidosis and dehydration and hyperglycemia) is same in ketoacidosis but is more severe. There is hypernatremia caused by renal sodium resorption in response to hypovolaemia, together with osmotic diuresis causing persistent free water loss. 

Non-ketotic hyperosmolar state usually occurs during marginal insulin deficiency, and their insulinaemia has sufficient antilipolytic effect to prevent the lipolytic and ketotic problems seen in ketoacidosis. There is decrease in anion gap <20 mmol/L and bicarbonate is normal and pH >7.30. There is hypernatraemia and more severe water loss 18 L in typical adult.

ALCOHOLIC KETOACIDOSIS

During alcoholism and resulting poor diet is association with vomiting, this cause ketoacidosis and low, normal or elevated blood glucose. Ketosis is caused by lack of insulin action which results in mobilization of NEFAs and their conversion to ketone bodies as alternative fuel. This is potentiated by counter-regulatory hormones like glucagon, cortisol and catecholamines secreted in response both to hypoglycaemia and extracellular fluid volume contraction. 

In addition, alcohol metabolism depletes cellular NAD⁺ which by restricting pyruvate formation from lactate, causes accumulation of lactate and depletion of pyruvate, a gluconeogenic substrate. As is the case in DKA, alteration in mitochondrial redox state favors beta hydroxybutyrate over acetoacetate production. A complex acid-base disorder ensues from the combined effects of ketosis causing metabolic acidosis, and a combination of extracellular fluid contraction and vomiting causing metabolic alkalosis. 

DIABETIC EMERGENCIES: DIABETIC KETOACIDOSIS (DKA)

DIABETIC KETOACIDOSIS

Approximately 30% of patient with type 1 diabetes present with ketoacidosis with clinical features of dehydration, shock, vomiting, abdominal pain, acidosis and cerebral impairment. There are four mechanisms of ketoacidosis: insulin deficiency, counter-regulatory hormone excess, fasting and dehydration. The most important is insulin deficiency. Hyperglycaemia and excess lipolysis cause dehydration and high circulating concentrations of NEFAs. Due to this hyperglycemia, ketosis and dehydration there is increase release in of counter-regulatory hormones which induce further hyperglycemia and lipolysis along with insulin resistance.

Biochemical features of ketoacidosis include hyperglycaemia, ketosis, metabolic acidosis and uremia. The characteristic ketosis is the consequence of increased lipolysis and decreased fat synthesis. Excess acetyl-CoA derived from beta oxidation of fatty acid is converted to the ketone bodies, acetoacetate and beta hydroxybutyrate with some acetone. Plasma beta hydroxybutyrate are 3 times more than acetoacetate.

Hyponatremia results from osmotic movement of intracellular water to interstitial and intravascular compartments drawn towards the hyperglycaemic plasma. Lipaemic serum (due to hypertriglyceridaemia) also gives false low sodium value.

Whole body potassium depletion is universal in DKA. Administration of insulin can also cause hypokalemia as insulin cause intracellular flux of potassium.

Cerebral oedema is one of the most feared complications of ketoacidosis mostly occurring in children. 

What is Somogyi effect and Dawn phenomenon ?

SOMOGYI EFFECT AND THE DAWN PHENOMENON

A special form of rebound from hypoglycaemia is the somogyi phenomenon, in which nocturnal hypoglycaemia occurs. There is awakening with malaise, headache and bedclothes damp from sweating are suggestive. Again due to falling blood glucose counter regulatory hormone are released and again hyperglycemia occurs. The rebound from the nocturnal hypoglycaemia results in patient waking with blood glucose concentration higher then desirable, causing the temptation to take at least as much (or even more) insulin the next night.

Non-diabetic subjects show circadian changes in blood glucose. The most marked such circadian effect is the dawn phenomenon which typically occurs between 4 and 7h and is an increase in plasma glucose and decrease in insulin sensitivity due to increased secretion of counter-regulatory hormones at that time. During this period people with diabetes usually experience modest rise (20-40 mg/dl) in blood glucose without ingestion of food.

Brittle diabetes

This is a condition of episodes of hypo or hyperglycaemia whatever their cause. Causes include psychological abnormalities such as eating disorders, personality disorders, etc. Other causes are inappropriate education, unsuitable insulin regimen, intercurrent illness such as thyroid disease, Addison’s disease, SLE (antibodies to insulin or its receptor), etc.

CONSEQUENCES OF DIABETES

Fig. Causes of Type 2 Diabetes

The risk of hypoglycaemia is the main limitation to achievement of good glycaemic control in diabetes.  In normal subjects the first response to falling blood glucose is reduction in insulin secretion occurring at blood glucose level below 80 mg/dl. This is lacking in subjects with type 1 diabetes or type 2 diabetes. Glucagon forms the next layer of defence, stimulating hepatic glycogenolysis and gluconeogenesis. However, most patients with type 1 and 2 are chronically hyperglucagonaemica and cannot respond to hypoglycaemia in this way. The last level of defence against acute hypoglycaemia is activation of the sympathetico-adrenal system, which normally occurs when blood glucose falls to below 55 mg/dl. This increases lipolysis and circulating NEFA (Non-esterified fatty acid) production and utilization, and mobilization of substrates for gluconeogenesis further inhibits insulin secretion and promotes glucagon release. Activation of the sympatheticopadrenal system gives first-clear symptoms of hypoglycaemia which is due to autonomic activation and Neuroglycopenia.  

Fig. Consequences of Diabetes

Clinical pseudohypoglycaemia and non-clinical pseudohypoglycaemia (which is a measure of low blood glucose due to severe leukocytosis or polycythemia). In clinical pseudohypoglycaemia patients with chronic hyperglycaemia experiences symptoms of hypoglycaemia. 

CLINICAL MANAGEMENT OF DIABETES MELLITUS

DIET

Dietary modifications includes,

• Low intake of simple carbohydrates with increase uptake in complex carbohydrates which can be slowly absorbed and have high glycemic index. Carbohydrates (complex carbohydrates) should provide approximately 55% of total energy
• Protein should provide 15% of total energy.
• There should be no more than 30% energy intake from fat, with increase in uptake of unsaturated fatty and <7% saturated fatty acid uptake.
• Sodium intake should not exceed 6g/day and plentiful fruits preferably less sugar containing and vegetables (five portions a day).
• A total daily dietary fiber intake of 40g is ideal.

EXERCISE

Regular low-intensity exercise like brisk walking, swimming or cycling for 30 min 3-5 times/week. This improves glucose disposal (by increasing GLUT4 in skeletal muscle), prevents progression from IGT to type 2 diabetes (by about 50%), increases basal metabolic rate (BMR) and reduces cardiovascular events.

SMOKING CESSATION

PHARMACOLOGICAL MANAGEMENT

ASPIRIN

Aspirin (or clopidogrel if aspirin is contraindicated) should be given for all men and women with type 1 or 2 diabetes over the age of 40, and those over 30 who have additional risk factors (e.g. family history, hypertension, smoking, dyslipidaemia, albuminuria). In lower age aspirin is avoided due to risk of Reye’s syndrome.

LIPID LOWERING AGENT

Consumption of saturated fat, cholesterol and transunsaturated fat, inadequate exercise are the primary cause of dyslipidaemia whereas alcohol excess, hypothyroidism, liver disease are the secondary cause of dyslipidaemia.

Metformin, pioglitazone and insulin can be used as lipid lowering agent; they either increase insulin action or reduce the flux of NEFA to liver (pioglitazone).

HMG-CoA reductase inhibitors, statins: They lower LDL-C. The ADA currently recommends an LDL-C target of 2.6 mmol/L in all patients over 40 years with diabetes as primary prevention, and in younger people with risk factors. ADA also recommends targets for tirglycerides of 1.7 mmol/L and HDL above 1.1 mmol/L.

HYPERTENSION

The ADA adopted the target of 130/80 to start treatment of hypertension in diabetes. ACE inhibitors of angiotensin II blockers are first line agents in diabetes. Amlodipine is the second line of drug in combination with ACE inhibitor Angiotensin receptor blocker, ARB.

ACE INHIBITORS AND ANGIOTENSIN II RECEPTOR ANTAGONIST

Angiotensin II (ATII) increases hepatic glucose production and decreases insulin sensitivity. Use of these agents increases insulin sensitivity. These are indicted for subjects with diabetes and hypertension, microalbuminuria, proteinuria, mild to moderate renal impairment, diabetic retinopathy, ischaemic heart disease and stroke.

HYPOGLYCAEMIC TREATMENT IN DIABETES

METFORMIN

This improves glycaemic control without weight gain. This is the first choice in treating type 2 diabetes especially overweight subjects. It reduces hepatic glucose output, improves peripheral glucose uptake and utilization in insulin-sensitive tissues (muscle, adipose tissue tissue) and reduces intestinal glucose transport. In type 2 diabetes, metformin can be used as monotherapy, or combined with insulin or with sulphonylureas and/or thiazolidinediones. In type 1 it is used with insulin for obese adults.

The main side effect of the use of biguanides (of which metformin is one) is lactic acidosis presented with lethargy nausea, vomiting, abdominal pain.

Biochemical features of lactic acidosis are elevated anion gap metabolic acidosis with high blood lactate.

SULPHONYLUREAS (AND RELATED INSULIN SECRETOGOGUES)

These drugs acts as insulin secretogogues, reducing glucose by augmenting the firs-phase insulin release.

In beta cells the APT dependent potassium channel has regulator domain of sulphonylurea receptor 1 (SUR-1). Sulphonylurea binds to this site and cause closure of K_ATP channels depolarizing the membrane, causing rapid influx of calcium ions via voltage dependent calcium channels. The resultant increase in free ionized calcium triggers cytoskeletal trafficking of secretory granules to plasma membrane and release of insulin by exocytosis. Other drugs like Glibenclamide, meglitinides nateglinide acts through same mechanism binding to SUR-1.

These drugs in contraindicated in type 1 diabetes, pregnancy, lactation and hepatic and renal insufficiency.

PPAR- γ ANALOGUES:

E.g. Thiazolidinediones; these are ligands for orphan nuclear peroxisome proliferator activator receptor family (PPARα, PPARγ, and PPARδ). These receptors are expressed in tissue that metabolizes fatty acids extensively like liver, kidneys, heart and muscle. They also increases HDL-C apolipoproteins, apo A-I, II decrease hepatic C-III production thus lowering TG vial reduced formation of VLDL. The nuclear PPAR receptors are endogenously activated by fatty acids and fatty acid-derived eicosanoids and the action of fibrate group of lipid lowering agents is mediated via PPARα receptors. Activation of PPARs leads to formation of heterodimers with the retinoid X receptor (RXR), bound to its own endogenous ligand, retinoic acid. These PPAR-RXR dimers bind to their response element (PPREs) modulating transcription of >40 target genes.

The insulin sensitizing effect of PPARγ agonist is due to fatty acid steal mechanism (i.e. changes in NEFA metabolism benefits for other tissues). These increases free fatty acid uptake in adipose tissue (by about 60%) and also increase fatty acid oxidation in liver, heart, kidneys and skeletal muscle. So, hepatic uptake of NEFA is reduced by 40%, rendering liver more insulin sensitive and giving these agents a potential role in treatment of hepatic steatosis. In adipose tissue they cause adipocyte differentiation and fat distribution from central to subcutaneous depots further reducing hepatic uptake of NEFA.

Thiazolidinediones are used in combination with both metformin and sulphonylurea as triple therapy. Other PPAR analogues are pioglitazone, rosiglitazones.

INSULINS 

In type 1 diabetes beta cell function, that falls to 10% of normal at disease presentation, doubles after initiation of insulin therapy and metabolic stabilization (honeymoon effect). This may be due to amelioration of glucotoxicity or lipotoxicity on the reduced numbers of and metabolically stressed beta cells.

All patients with type 1 diabetes are treated with exogenous insulin. Both long-acting or basal and short-acting or bolus insulin are used. Rapid acting insulin has rapid onset of action (<15 min), permitting injection immediately before or just after eating and has 3-5 hours of action which reduces the risk of hypoglycemia before next meal. It has sharper peak response resembling first-phase insulin release in normal persons. Some rapid acting insulins are Insulin aspart, it is homologous to human insulin with exception of single substitution of aspartate for proline in position B28. In Insulin lispro proline and lysine at B28 and 29 respectively are reversed.

Long acting insulins e.g. glargine and detemir, has 24h duration of action with minimum peak action. The regimen consists of twice daily insulin mixture of longer and shorter acting insulins in ratio typically between 75/25 and 60/40. Another compromise of single dose of long-acting insulin at night with doses of short-acting insulin immediately before meals during the day (basal-bolus regimen).

In type 2 diabetes patients require insulin treatment after a median of 7 years from diagnosis. Insulin treatment in overweight or obese has risk of further weight gain, which increase the need for escalating insulin does and spiraling obesity. Reasons for weight gain after starting insulin in type 2 diabetes include a reduction in energy wastage through glycosuria, anabolic effects of insulin, reduction in attention to diet and exercise in presence of an highly effective means of glycemic control and increased eating because of the need to avoid or treat hypoglycemia on insulin regimens. These patients do not require exogenous insulin throughout 24 hours. Most patients with type 2 diabetes especially those who are overweight, should remain on metformin when insulin is instituted in whatever form.  

URINARY ALBUMIN EXCRETION (UAE) : INTRODUCTION AND ITS IMPLICATIONS

Patients with diabetes mellitus are at high risk of suffering renal damage. Diabetes is the most common cause of end -stage renal disease (ESRD). Although nephropathy is less common in patients with type 2 diabetes, approximately 60% of all cases of diabetic nephropathy occur in these patients because of the higher incidence of this form of diabetes. Early detection of diabetic nephropathy relies on tests of urinary excretion of albumin. Persistent proteinuria detectable by routine screening tests (equivalent to a urinary albumin excretion [UAE] rate greater than or equal to 30 mg/d) indicates overt diabetic nephropathy. Once diabetic nephropathy occurs, renal function deteriorates rapidly and renal insufficiency evolves. Treatment at this stage can retard the rate of progression but not stop or reverse the renal damage. Preceding this stage is a period of increased UAE not detected by routine methods. This range of 20 to 200 μg/min (or 30 to 300mg/24hr or albumin/creatinine ratio of 30-300 μg/mg) of increased UAE defines microalbuminuria. Note that it is not defined in terms of urinary albumin concentration, although the albumin: creatinine ratio can be used as a substitute for albumin measurements in a time collection of urine. The term microalbuminuria implies a small version of the albumin molecule rather than an excretion rate of albumin greater than normal but less than that detectable by routine methods. Clinical proteinuria or microalbuminuria is established with an albumin-creatinine ratio of ≥300 μg/mg or protein excretion ≥300 mg/day. 

The presence of increased UAE denotes an increase in the transcapillary escape rate of albumin and is therefore a marker of microvascular disease. Persistent UAE greater than 30 mg/d represents a twentyfold greater risk for the development of clinically overt renal disease in patients with type 1 and type 2 diabetes. Prospective studies have demonstrated that increased UAE precedes and is highly predictive of diabetic nephropathy, end-stage renal disease, cardiovascular mortality, and total mortality in patients with diabetes mellitus. In addition increase UAE identifies a group of nondiabetic subject at increased risk of coronary artery disease.

UAE is increased by physiological factors (e.g., exercise, posture, and diuresis) and the method of urine collection must be standardized. Samples should not be collected after exertion, in the presence of urinary tract infection, during acute illness, immediately after surgery, or after an acute fluid load. All the following urine samples are currently acceptable: 

(1) 24-hour collection; 

(2) overnight (8 to 12 hours, timed) collection; 

(3) 1- to 2-hour timed collection (in laboratory or Clinic); or 

(4) first morning sample for simultaneous albumin and creatinine measurement. 

Only results for timed specimens can be reported as mg albumin excreted per hour, but the albumin: creatinine ratio is more practical and convenient for the patient and is the recommended method. A first morning void sample is best because it has a lower within-person variation for the albumin: creatinine ratio than a random urine sample. At least three separate specimens, collected on different days, should be assayed because of the high intraindividual variation diurnal variation (50% to 100% higher during the day). Urine should be stored at 4⁰C after collection. Alternatively, 2 mL of 50 gm/L sodium azide can be added per 500 mL of urine, but preservatives are not recommended for some assays. Bacterial contamination and glucose have no effect. Specimens are stable for 2 weeks at 4 'C and for at least 5 months at -80⁰C. Albumin concentration decreased by 0.27% at -20⁰C. Freezing samples has been reported to decrease albumin, but mixing immediately before assay eliminates this effect.

The test strips most of which are optimized to read positive at predetermined albumin concentration have been recommended for screening programs. Test strips contains bromophenol blue in alkaline matrix to detect albumin concentrations exceeding 40 mg/L. Other test strips include antialbumin IgG complexed to galactosidase. The albumin in the urine binds to antibody enzyme conjugate in the test strip. Excess conjugate is retained in a separate zone containing immobilized albumin and only albumin bound to the antibody-enzyme immunocomplex diffuses to the reaction zone. Here it reacts with a buffered substrate (chlororphenol red galactoside) to produce a red color when the beta galactosidase hydrolyzes galactose.

For quantitation different RIA, ELISA radial immunodiffusion and immunoturbidimetry are available.

The ADA recommends initial UAE measurement in type 1 diabetes patients who have had diabetes more than or equal to 5 years and in all type 2 diabetic patients. Because of the difficulty in dating the onset of type 2 diabetes, screening should commence at diagnosis. Analysis should be performed annually in all patients who have a negative screening results. If screening result is positive UAE should be evaluated by quantitative assay. Diagnosis requires the demonstration of increased UAE in at least two of 3 tests measured within 6 month period.

(Source: Tietz Clinical Chemistry, 4th Edition)

TEST FOR INSULIN RESISTANCE

Subjects requiring large amount of insulin to maintain euglycaemia e.g. >150 units or 1.5 units/kg body weight/day, insulin resistance may be postulated. For this insulin is administered intravenously and subcutaneously and the level of glucose and insulin in plasma is measured. Normal fasting insulin concentration are up to 20 mU/L. Hyperinsulinaemic clamp is the reference measure of insulin resistance. In euglycaemic variant of the test, insulin is infused into a peripheral vein so as to raise the plasma insulin concentration to a target range around 60 mU/L. 

The plasma glucose concentration is measured every 5-10 min and glucose is infused peripherally to maintain glucose concentraions within the desired range. When a steady state has been reached (usually 90-120 min), the rate of exogenous glucose infusion needed to maintain the glucose concentration is an index of the glucose clearance rate and of the subject’s insulin sensitivity.

Glucose transporter function can be assayed by incubating cells of interest (e.g. leukocytes, monocytes, adipocytes) with a non-metabolizable glucose analog such as 2-deoxyglucose. The cellular content of the glucose analogue after a given time provides a measure of glucose transporter function.  

MEASUREMENT OF β- CELL FUNCTION

Measurement of plasma C-peptide concentration can be done. Elevated fasting plasma proinsulin indicates subjects with abnormal beta cell function, even if glucose tolerance is normal. 

ADVANCED GLYCATION END PRODUCTS: AN INTRODUCTION

The molecular mechanism by which hyperglycemia produces toxic effect is unknown, but glycation of tissue proteins may be important. Nonenzymatic attachment of glucose to long lived proteins like collagen or DNA, produces stable Amadori early Glycated products. These undergo a series of additional rearrangements dehydration and fragmentation reactions, resulting in stable advanced glycation end products (AGE). The amounts of these products do not return to normal when hyperglycemia is corrected and they accumulate continuously over the lifespan of the protein. Hyperglycemia accelerates the formation of protein-bound AGE, and patients with diabetes mellitus thus have more AGE than healthy subjects. Through effects on the functional properties of protein and extracellular matrix, AGE may contribute to the microvascular and macrovascular complications of diabetes mellitus. Moreover an inhibitor of AGE formation, aminoguanidine has been shown to prevent several complications of diabetes in animal model.

In healthy people Hb-AGE accounts for 0.4% of circulating Hb, with significantly higher in diabetes mellitus. After acute change in glycemia, Hb-AGE level changes, but the rate of alteration is 23% slower than that of HbA_1c. Thus Hb-AGE provides a measure of diabetic control longer than that indicated by GHb, reflecting blood glucose concentration over a greater proportion of life of red blood cells. 

Fructosamine and it's implications

FRUCTOSAMINE

Fructosamine is a ketoamine product of protein glycation formed when glucose bound to variety of proteins by aldimine linkage undergoes an Amadori rearrangement. The major component of fructosamine in plasma is Glycated albumin. Fructosamine is easily measured (using nitroblue tetrazolium assay); its concentration reflects control over the preceding 15-20 days. When the patient has abnormal hemoglobins, or during pregnancy alternative tests should be used. Glycated albumin and Glycated fibrinogen are proposed for such conditions. Albumin has half life of approximately 20 days, so fraction that is Glycated reflects glycaemic control for the preceding 1-2 weeks.

(Source: Tietz Clinical Chemistry, 4th Edition)

Fructosamine is the generic name for plasma protein ketoamine. There is interaction of glucose with the ε-amino group on lysine residue of albumin. Because all Glycated serum proteins are fuctosamines and albumin is the most abundant serum protein, measurement of fructosamine is thought to be largely a measure of Glycated albumin. As fructosamine determination monitors short term glycemic changes different from GHb, it may have a role in conjunction with GHb rather than instead of it. In addition fructosamine may be useful in patients with hemoglobin variants such as HbS or HbC that are associated with decreased erythrocyte lifespan where GHb is of little value. Fructosamine values are highly affected and not recommended in conditions that affect protein turnover like liver cirrhosis, nephrotic syndrome or dysproteinemias, inflammatory conditions. It is generally accepted that the test should not be performed when serum albumin is less than 30g/L.

 

Methods for measuring Glycated proteins include affinity chromatography using immobilized phenylboronic acid, HPLC of Glycated lysine residue after hydrolysis of Glycated proteins, photometric procedure in which mild acid hydrolysis releases 5-hydroxymethylfurfural- proteins are precipitated with TCA and the supernatant is reacted with 2-thiobarbituric acid; and other procedures using phenylhydrazine and furosine.  Another method is under alkaline conditions which results in fructosamine undergoing an Amadori rearrangement and the resultant compounds having reducing activity that can be differentiated from other reducing substances. In the presence of carbonate buffer, fructosamine rearranges to the eneaminol form, which reduces NBT to a formazan. The absorbance at 530 nm is measured at two time points and the absorbance change is proportional to the fructosamine concentration. 

TEST FOR RECENT GLYCAEMIC CONTROL: HbA1c measurement

MEASUREMENT OF GLYCATED HEMOGLOBIN

Glycation is the non enzymatic addition of sugar residue to amino groups of proteins. In adults HbA constitute the major fraction (97%) also has other subforms namely A_1a, A_1b, A_1c which are collectively called HbA₁, fast hemoglobins, glycohemoglobins or Glycated hemoglobins. HbA_1c is formed by the condensation of glucose with N-terminal valine residue of each β-chain of HbA to form an unstable Schiff base (aldimine, pre-HbA_1c). The Schiff base may either dissociate or undergo an Amadori rearrangement to form a stable ketoamine, HbA_1c. HbA_{1a1, 1a2} which make up HbA_1a have fructose-1, 6-diphosphate and glucose-6-phosphate, respectively attached to amino terminal of the β-chain. Other are HbA_1b has pyruvate attached to N-terminal of beta chain.  HbA_1c is the major fraction constituting approximately 80% of HbA₁.

Glycation may also occur at sites other than the end of beta chain, such as lysine residue or the alpha chain. These GHbs referred to as Glycated HbA₀ or total Glycated Hb. These are measured by boronate affinity chromatography.

(Source: Tietz Clinical Chemistry, 4th Edition)

Formation of GHb is essentially irreversible and the concentration in the blood depends on both the lifespan of the red blood cell (average 120 days) and the blood glucose concentration. Since erythrocyte is free permeable to glucose. Because the rate of formation of GHb is directly proportional to the concentration of glucose in the blood, the GHb concentration represents the integrated values for glucose over the preceding 6 to 8 weeks. This provides an additional criterion for assessing glucose control because GHb values are free of day to day glucose fluctuations and are unaffected by recent exercise or food ingestion.

The interpretation of GHb depends on the red blood cells having a normal lifespan. Patients with hemolytic disease or other conditions with shortened red blood cells survival exhibit a substantial reduction in GHb. Similarly individuals with recent significant blood loss have false low values owing to higher fraction of young erythrocytes. High GHb concentrations have been reported in iron deficiency anemia, probably because of high proportion of old erythrocytes. Presence of other hemoglobinopathies can alter results. Presence of carbamylated Hb which is formed by attachment of urea and is present in large amount in renal failure and common in diabetic patients, also produce altered results.

GHb has been established as an index of long term blood glucose concentration and as a measure of the risk for the development of complications in patients with diabetes mellitus. There is direct relationship between blood glucose concentration (assessed by HbA_1c) and the risk of complications. The absolute risks of retinopathy and nephropathy were directly proportional to the mean HbA_1c. Studies have shown reduction in HbA_1c level will significantly reduce the risk of microvascular complications and retinopathy and nephropathy and cardiovascular disease. ADA recommends that a primary treatment goal in adults with diabetes should be near normal glycemia with HbA_1c <7%. HbA1c of 7% (of total HbA) corresponds with mean plasma glucose of approximately 170 mg/dl, and each 1% increase with a 36 mg/dl increase in mean plasma glucose concentrations.

There are more than 30 different methods for determination of GHbs. These methods separate hemoglobin from GHb using technique based on charge differences (ion-exchange chromatography, HPLC, electrophoresis, IEF), structural differences (affinity chromatography and immunoassay), or chemical analysis (photometry and spectrophotometry). The result in all is expressed as percentage of total Hb.

Ion exchange chromatography separates Hb variants on the basis of charge. The cation exchange resin (negatively charged) packed in disposable minicolumn has an affinity for Hb, which is positively charged. The patient’s sample is hemolyzed and an aliquot of the hemolysate is applied to the column. A buffer is applied and the eluent collected. Here GHb is less positively charged than other so will elute first than other. The eluted GHb (A1a, 1b and 1c, collectively A1) are measured in spectrophotometer. Other Hbs are also measured after subsequent elution and the HbA1 is expressed as percentage of total.

HPLC can be used for separation and quantitation of HbA1c and other fractions. HPLC employs, cation exchange chromatography.

Agar gel electrophoresis on whole blood hemolysates at pH 6.3 provides good resolution of HbA and HbA₁. The gel contains negatively charged moieties that interacts with the hemoglobin. After 25 to 35 minutes, the GHb separates on the cathodic side of HbA. Quantification is done by scanning densitometry at 415 nm.

The hemoglobin variant separate on IEF on the basis of their migration in gel containing pH gradient on acrylamide gel slabs.

Immunoassay with the principle of immunoinhibition are used like ELISA where antibodies are raised and used to inhibit other fraction in one hand and capture and detection antibodies are used to determine HbA1c.

Affinity gel columns are used to separate GHb, which binds to the column, from the nonglycated fraction. M-Aminophenylboronic acid is immobilized by cross linking to beaded agarose or another matrix (e.g., glass fiber). The boronic acid reacts the cis-diol groups of glucose bound to Hb to form a reversible five member ring complex thus selectively holding the GHb on the column. The nonglycated Hb does not bind. Sorbitol is then added to elute the GHb. Absorbance of the bound and nonbound fractions measured at 415 nm is used to calculate the percentage of GHb. Nonglycated Hb does not bind and is removed In a wash step. The sorbitol competes for boronate binding sites. 

(Source: Tietz Clinical Chemistry, 4th Edition)

For borate affinity assay, packed blood cells are mixed with hemolysate reagent that contain borate buffer. Glycated Hb is assayed from this hemolysate.