VIOLATIONS OF CARBOHYDRATE METABOLISM.
LABORATORY DIAGNOSTICS OF DIABETES MELLITUS.
UrINE ANALYSIS.
Diabetes is the most common metabolic disorder, and its incidence is increasing. Biochemical measurements are particularly important in detecting it, monitoring its control and treating its metabolic complications. Hypoglycaemia occurs in insulin-treated diabetic patients, but is otherwise rare. However, it is an important diagnosis to make because of its possible consequences. Other disorders of carbohydrate metabolism are uncommon.
Dietary carbohydrate is digested in the gastrointestinal tract to simple monosaccharides which are then absorbed. Starch provides glucose directly,while fructose (from dietary sucrose) and galactose (from dietary lactose) are absorbed and also converted into glucose in the liver. Glucose is the common carbohydrate currency in the body.
Insulin is the principal hormone affecting blood glucose levels. It is a small protein synthesized in the beta cells of the islets of Langerhans of the pancreas. It acts through membrane receptors and its main target tissues are liver, muscle and adipose tissue. the overall effects of insulin is to promote cellular uptake and storage of metabolic fuels. Insulin is synthesised in the ß-cells of the islets of Langerhans in the pancreas. It is formed as prepro-insulin, which is rapidly cleaved to pro-insulin. The pro-insulin is packaged into secretory granules in the Golgi apparatus and cleaved to insulin and C peptide. Insulin and C peptide are later released into the circulation in equimolar amounts. A rise in blood [glucose] is the main stimulus for insulin secretion. Some amino acids (e.g. leucine), fatty acids and ketone bodies also promote insulin secretion. The release of insulin in response to hyperglycaemia is enhanced by the presence of GIP or glucagon. GIP is probably the most important factor in the larger release of insulin that occurs in response to an oral glucose load, compared with the same dose of glucose given intravenously. Vagal stimulation also promotes insulin release.The insulin receptor is located on the cell surface and is internalised after insulin binding. Within different organs, target enzymes have been identified that serve to explain the known effects of insulin on intermediary metabolism. For instance, activation of glucose transport, induction of hexokinase (or glucokinase) and activation of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in the liver are all consistent with the actions insulin in promoting increased glucose uptake and glycolytic breakdown. Stimulation of glycogen synthase accords with the effects of insulin on glycogen formation in the liver.
The effects of insulin are opposed by other hormones, glucagon, adrenaline, glucocorticoids and growth hormone. The blood glucose concentration is the result of a balance between these different endocrine forces.
Diabetes mellitus is a group of diseases characterized by an elevated blood glucose level (hyperglycaemia) resulting from defects in insulin secretion, in insulin action, or both. Diabetes mellitus is not a pathogenic entity but a group of aetiologically different metabolic defects.
Common symptoms of diabetes are lethargy from marked hyperglycaemia, polyuria, polydipsia, weight loss, blurred vision and susceptibility to certain infections. Severe hyperglycaemia may lead to hyperosmolar syndrome and insulin deficiency to life-threatening ketoacidosis. Chronic hyperglycaemia causes long-term damage, dysfunction and failures of various cells, tissues and organs.
Classification of diabetes mellitus:
1. Type 1 diabetes mellitus
Immune mediated
Idiopathic
2. Type 2 diabetes mellitus
3. Other specific types of diabetes
Genetic defects of islet ß-cell function
Genetic defects of insulin action
Diseases of the exocrine pancreas
Endocrinopathies
Drug- or chemical- induced diabetes
Infections
Uncommon forms of diabetes
Other genetic syndromes
4. Gestational diabetes mellitus
Primary diabetes mellitus is subclassified into insulin dependent diabetes mellitus (IDDM or type I) and non- insulin dependent diabetes mellitus (NIDDM or type II).
The contrasting features of IDDM and NIDDM are shown in Table 1:
Secondary diabetes mellitus may result from pancreatic disease, endocrine disease such as Cushing’s syndrome, drug therapy, and insulin receptor abnormalities.
Insulin dependent diabetes mellitus (IDDM or type I) accounts for approximately 15 % of all diabetics. It can occur at any age, but is most common in the young, with a peak incidence between 9 and 14 years of age. The absolute lack of insulin is a consequence of the autoimmune destruction of insulin-producing beta cells.
Markers:
__ islet cell antibodies (ICAs)
__ auto-antibodies to insulin (IAAs)
__ auto-antibodies to glutamic acid decarboxylase (GAD65)
__ auto-antibodies to tyrosine phosphatases IA-2 and IA-2ß
Laboratory findings:
__ Hyperglycaemia
__ Ketonuria
__ Low or undetectable serum insulin and C-peptide levels
__ Auto-antibodies against components of the islet ß-cells
Non- insulin dependent diabetes mellitus (NIDDM or type II) accounts for approximately 85 % of all diabetics and can occur at any age.It is most common between 40 and 80 years. In this condition there is resistance of periferal tissues to the actions of insulin, so that the insulin level may be normal or even high. Obesity is the most commonly associated clinical feature.
Type 2 diabetes is due to insulin insensitivity combined with a failure of insulin secretion to overcome this by hypersecretion, resulting in relative insulin deficiency. There is a strong genetic predisposition. Type 2 diabetes is more common in individuals with family history of the disease, in
individuals with hypertension or dyslipidaemia and in certain ethnic groups.
The risk of developing Type 2 diabetes increases with:
– Family history of diabetes (in particular parents or siblings with diabetes)
– Obesity (≥ 20% over ideal body weight or BMI ≥ 25.0 kg/m²)
– Membership of some ethnic groups
– Age ≥ 45 years
– Previously identified IFG or IGT
– Hypertension (≥ 140/90 mmHg in adults)
– HDL cholesterol level <1.0 mmol/L (<0.38 g/L) and/or a triglyceride level ≥ 2,3 mmol/L (≥2,0 g/L)
– Reduced physical activity
– History of gestational diabetes mellitus (GDM) or delivery of babies >
Laboratory findings:
__ hyperglycaemia
__ hyperlipidaemia
__ high serum insulin/C-peptide level
__ defective insulin secretion
__ insulin resistance
Late complications of diabetes mellitus
Diabetes mellitus is not only characterized by the presence of hyperglycemia but also by the occurrence of late complications:
1. Microangiopathy is defined as abnormalities in the walls of small blood vessels, the most prominent feature of which is thickening of the basement membrane.
2. Retinopathy may lead to blindness because of vitreous haemorrhage from proliferating retinal vessels, and, maculopathy as a result of exudates from vessels or oedema affecting the macula.
3. Nephropathy leads ultimately to renal failure. In the early stage there is kidney hyperfunction, associated with an increased GFR, increased glomerular size and microalbuminuria. In the late stage, there is increasing proteinuria and a marked decline in renal function, resulting in uraemia.
4. Neuropathy may become evident as diarrhoea, postural hypotension, impotence, neurogenic bladder and neuropathic foot ulcers due to microangiopathy of nerve blood vessels and abnormal glucose metabolism ierve cells.
5. Macroangiopathy leads to premature coronary heart disease. The exact mechanisms for increased susceptibility to atherosclerosis.
Clinical symptoms of hyperglycemia include:
· polyuria
· polydipsia
· lassitude
· weight loss
· pruritus vulvae
· balanitis
These symptoms are common to both NIDDM and IDDM but are more pronounced in IDDM. It is important to remember that patients with NIDDM may be completely asymptomatic.
Gestational diabetes mellitus (GDM)
Definition: Any degree of clinical glucose intolerance with onset or first recognition during pregnancy.
GDM complicates the pregnancy: The following problems may develop with GDM:
altered duration of pregnancy
placental failure
hypertension / pre-eclampsia
high birth weight of the newborn
Therapy:
nutrition therapy
insulin (glucose-lowering drugs not advised).
Diagnosis of GDM:
Fasting plasma glucose level >7,0 mmol/L (>1,26 g/L) or casual plasma glucose
>11,1 mmol/L (>2,00 g/L), confirmed on a subsequent day.
Laboratory strategy to diagnose GDM:
One step approach: OGTT (
Two step approach: 1. First OGTT with
2. Second OGTT with
Six weeks after pregnancy or later the woman should be re-examined for the presence of diabetes mellitus or IGT.
Screening for diabetes
Screening for diabetes is an analytical, organizational, and financial challenge.
There are two strategies that may be applied for screening
1. Detect all people with diabetes in a population.
2. Detect diabetes amongst those people who are mostly likely to have diabetes (selective screening)
Selective screening should consider individuals :
__ with typical symptoms of diabetes
__ with a first-degree relative with diabetes
__ who are members of a high risk ethnic group
__ who are overweight (BMI ≥ 25.0 kg/m²)
__ who have delivered a baby >
__ who are hypertensive (≥ 140/90 mmHg)
__ with raised serum triglyceride and cholesterol levels
__ who were previously found to have IGT or IFG
The basic laboratory measures for screening are:
1. Fasting capillary blood glucose
2. Glucosuria
3. HbA1c (Glycated hemoglobin)
4. OGTT (Oral glucose tolerance test)
The common and best indicator for estimating diabetes prevalence and incidence is fasting blood glucose (FPG). FPG concentration. of >7,0 mmol/L (>1,26 g/L) is an indication for retesting. For centralized screening the analysis of glycated haemoglobin (HbA1c) from a blood drop is recommended, though this approach is more expensive than FPG.
Fig. 1: Specimen collection device for centralized analysis of HbA1c
HbA1c blood carrier
labelling of transporting envelop
finger prick – capillary blood
drying of sample (approx 30 minutes)
sealing of the envelope and sending to the laboratory
Diagnosis and monitoring of diabetes mellitus
The diagnosis of diabetes mellitus has serious consequences. It confers a risk of long-term diabetic complications, including blindness, renal failure and amputations, as well as an increased risk of cardiovascular disease. It also means a lifetime of dietary restriction and medications and can seriously curtail lifestyle and employment prospects. The diagnosis may be suggested by the patient’s history, or by the results of dipstick tests for glucose on urine specimens. However, urine glucose measurements by themselves are inadequate for diagnosing diabetes. They potentially yield false-positive results in subjects with a low renal threshold for glucose, and in a patient with diabetes, they may yield false-negative results if the patient is fasting. A provisional diagnosis of diabetes mellitus must always be confirmed by glucose measurements on blood specimens.
The most recent criteria for the diagnosis of diabetes mellitus have been laid down by the World Health Organization (WHO) in 1998, and by the American Diabetes Association in 1997. These are broadly similar but differ in some details. It is likely that the precise requirements for the diagnosis of diabetes and the states of impaired glucose regulation will continue to evolve as knowledge of the relation between glucose regulation and the future development of complications accumulates. The following descriptions adhere to the WHO recommendations. Separate criteria are described depending on whether venous or capillary whole blood, or venous or capillary plasma specimens are used. In practice, results for this important diagnosis will usually come from a clinical laboratory and use venous plasma. According to the criteria, a random venous plasma [glucose] of 11.1 mmol/L or more, or a fasting plasma [glucose] of 7.0 mmol/L or more, establishes the diagnosis. A single result is sufficient in the presence of typical hyperglycaemic symptoms of thirst and polyuria. In their absence, a venous plasma [glucose] in the diabetic range should be detected on at least two separate occasions on different days. Where there is any doubt, an OGTT should be performed, and if the fasting or random values are not diagnostic, the 2-h value should be used. In practice, the diagnosis is often obvious clinically, and [glucose] is only needed for confirmation and is unequivocally high. The diagnosis should never be made on the basis of a single test in a patient without symptoms.
At present a raised HbA1c should not be used to make a diagnosis of diabetes. Standardisation of HbA1c analysis has been a problem in the past although this is now less of an issue. This has meant that it has not been possible to reliably determine a single cut-off level that would diagnose diabetes in all laboratories.
Glucosuria allows for a good first-line screening test for diabetes mellitus; normally glucose does not appear in the urine until the plasma glucose rises above 10 mmol/l (renal threshold for glucose – the maximum amount of glucose in blood, which is completely reabsorbed in kidney tubuls).
Ketone bodies may accumulate in the plasma of a diabetic patient. Ketones may be present in a normal subject as a result of simple prolonged fasting. Dry reagent strips which detect acetoacetate might therefore provide an understimate of ketonaemia/ketonuria.
Blood glucose
Glucose is routinely measured in the laboratory on blood specimen which have been collected into tubes containing fluoride, an inhibitor of glycolysis. Becouse of the need sometimes to obtain rapid blood glucose results and the wide-spread self-monitoring of diabetic patients, blood glucose is also assesed outside the laboratory using test strips.
The World Health Organization has published guidelines for the diagnosis of diabetes mellitus on the basis of blood glucose results and the response to an oral glucose load.
Random blood glucose (RBG) is the only test required in an emergency. An RBG of less than 8 mmol/l should be expected ion-diabetics.
RBG higher than 11 mmol/l usually indicates diabetes mellitus.
Fasting blood glucose (FBG) is measured after overnight fast (at least 10 hours). An FBG is better than RBG for diagnostic purposes. Ion-diabetics it is usually lower than 6 mmol/l. Fasting values of 6-8 mmol/l should be interpreted as borderline.
Oral glucose tolerance test (OGTT)
The OGTT is a provocation test to examine the efficiency of the body to metabolise glucose. The OGTT provides information on latent diabetes states. The OGTT distinguishes metabolically healthy individuals from people with impaired glucose tolerance and those with diabetes. The OGTT is more sensitive than FPG for the diagnosis of diabetes.
Preparation of the patient:
Three days unrestricted, carbohydrate rich diet and activity.
No medication on the day of the test.
12-h fast.
No smoking.
Glucose load: Adults
Children: 1,75 g/Kg up to
Plasma glucose levels are measured every 30 min for 2 hours.
Urine may also be tested for glucose at time 0 and after 2 hours. The patient should be sitting comfortably throughout the test, should not smoke or exercise and should have been oormal diet for at least 3 days prior to the test.
Indications:
· borderline fasting or post-prandial blood glucose
· persistent glycosuria
· glycosuria in pregnant women
· pregnant women with a family history of diabetes mellitus and those who previously had large babies or unexplained fetal loss
Interpretation of an OGTT
If the patient has a normal fasting plasma glucose and only the 2 h value in the diabetic range, the test should be repeated after approximately 6 weeks. Impaired glucose tolerance (IGT) should not be regarded as a disease. It signals that the patient is at an intermediate stage betweeormality and diabetes mellitus and is at an increased risk of developing diabetes. Such patients should be followed yearly, and dietary treatment may be used.
The OGTT is affected by metabolic stress from a number of clinical conditions and drug treatments, such as:
Major surgery
Myocardial infarction, stroke, infections, etc
Malabsorption
Drugs (steroids, thiazides, phenytoin, oestrogens, thyroxine)
Stress, nausea
Caffeine, smoking
Long-term indices of diabetes control:
A high concentration of glucose in the ECF leads to its non-enzymatic attachment to the lysine residues of a variety of proteins. This is called glycation. The extent of this process depends on the ambient glucose level . The concentration of glycated protein is therefore a reflection of a mean blood glucose level prevailing in the extracellular fluid during the life of that protein.
1. Haemoglobin A1c or glycated haemoglobin
The life span of haemoglobin in vivo is 90 to120 days. During this time glycated haemoglobin A forms, being the ketoamine compound formed by combination of haemoglobin A and glucose.
Several subfractions of glycated haemoglobins have been isolated. Of these, glycated haemoglobin A fraction HbA1c is of most interest serving as a retrospective indicator of the average glucose concentration over the previous 8 to 10 weeks.
The reaction of the non-enzymatic glycation of proteins is as follows:
There are a variety of commercial tests systems for measuring HbA1c. The majority of commercial tests separate HbA1c from non-glycated haemoglobin by chromatography. HbA1c can also directly be measured in blood by immuno-chemical techniques without being separated from nonglycated haemoglobin. While it is true that there is no biochemical interference from haemoglobin variants for the affinity and immunochemical methods, there may be a biological interference in certain conditions where the haemoglobin (erythrocyte) turnover in the blood is high.
Specimen: Whole blood is used for analysis.
Blood+EDTA 100 µl
Heparinized blood 100 µl
Capillary blood one drop on special filter paper
The specimen should be analyzed as soon as possible. In haemolysates adducts of haemoglobin with glutathione may be formed.
Relationship between HbA1c (DCCT standardized or equivalent) and average plasma or whole blood glucose concentrations from 7-point self-monitored profiles
HbA1c glucose (mmol/L)
(%) plasma blood
–––––––––––––––––––––––––
4,0 3,6 2,6
5,0 5,6 4,5
6,0 7,6 6,3
7,0 9.6 8,2
8,0 11,5 10,0
9,0 13,5 11,8
10,0 15,5 13,7
11,0 17,5 15,6
12,0 19,5 17,4
2. Fructosamine
Many other proteins in addition to haemoglobin are glycated when exposed to glucose in the blood. As indication of the extent of this glycation can be obtained by measuring fructosamine, the ketoamine product of non-enzymatic glycation. As albumin is the most abundant plasma protein, glycated albumin is the major contributor to serum fructosamine measurements. As this protein has a shorter half-life than haemoglobin, fructosamine measurements are complementary to HbA1c providing the index of glucose control over the 3 weeks prior to its measurement. Albumin is the main component of plasma proteins. As albumin also contains free amino groups, non-enzymatic reaction with glucose in plasma occurs. Therefore glycated albumin can similarly
serve as a marker to monitor blood glucose. Glycated albumin is usually taken to provide a retrospective measure of average blood glucose concentration over a period of 1 to 3 weeks.
Under alkaline conditions (pH: 10.35) glycated proteins (ketoamine) reduce nitroblue tetrazolium (NBT) to formazane. In the fructosamine test the absorption of formazane at 530 nm is photometrically measured and compared with appropriate standards to determine the concentration of glycated proteins in plasma, the major part being contributed by albumin.
Reference interval: 205- 285 µmol/L
Microalbuminuria
Diabetic patients are at high risk of developing renal insufficiency years after the onset of diabetes.
Diabetes is the most common cause of renal failure. In one third of patients with Type 1 diabetes diabetic nephropathy leads to end-stage renal disease requiring dialysis. In Type 2 diabetes renal failure is less frequent due to earlier death from vascular disease, but, since this type of diabetes is more prevalent, about half of the cases of diabetic nephropathy occur in these patients. The early signs of diabetic nephropathy cannot be detected by the routine screening tests for proteinuria, so that more sensitive methods for detecting abnormal albumin excretion must be used. The early stage of albuminuria is clinically defined as an albumin excretion rate of 30-300 mg/24 hours (20-200 µg/min), although true normal renal albumin excretion is lower than this. The small amount of albumin secreted in urine in early diabetic renal disease led to the misleading term “microalbuminuria”, which is still widely used but should be avoided. Raised albumin excretion rate is a cardiovascular risk factor in people with Type 2 diabetes (and indeed in the non-diabetic population), in whom it should be regarded as a predictor of both increased macro- and microvascular risk.
Diabetic ketoacidosis
All metabolic disturbances seen in DKA are the indirect or direct consequences of the lack of insulin. Decreased glucose transport into tissues leads to hyperglycemia which gives rise to glycosuria. Increased lipolysis causes over-production of fatty acids, some of which are converted into ketones, giving ketonaemia, metabolic acidosis and ketonuria. Glucosuria causes an osmotic diuresis, which leads to the loss of water and electrolytes – sodium, potassium, calcium, magnesium, phosphate and chloride. Dehydration, if severe, produces pre-renal uraemia, and may lead to hypovolaemic shock. The severe metabolic acidosis is partially compensated by an increased ventilation rate (Kussmaul breathing). Frequent vomiting is also usually present and accentuates the loss of water and electrolytes.
Laboratory investigations
Urine (if available) should be tested for glucose and ketones, and blood checked for glucose using a test strip. Venous blood should be sent to the laboratory for plasma glucose and serum sodium, potassium, chloride, bicarbonate, urea and creatinin. An arterial blood sample should also be sent for measurement of blood gases. Blood glucose should be monitored hourly at the bedsite until less than 15 mmol/l. Thereafter checks may continue 2-hourly. The plasma glucose should be confirmed in the laboratory every 2-4 hours.
Treatment
The management of DKA requires the administration of 3 agents:
· Fluids. Patients with DKA are usually severely fluid depleted and it is essential to expand their ECF with saline to restore their circulation.
· Insulin. Intravenous insulin is most commonly used.
· Potassium.
Hypoglycemia is a laboratory diagnosis which is usually taken to mean a blood glucose level below 2.5 mmol/l.
A low blood glucose level normally leads to the stimulation of catecholamine secretion and stimulation of glucagon, cortisol and grows hormone.
Symptoms mosr commonly seen in hypoglycaemia:
· sweating
· shaking
· tachycardia
· nausea
· weakness
·
Laboratory investigation
Blood glucose. The detection of hypoglycaemia is by blood glucose testing. Urine testing cannot detect hypoglycaemia.
Plasma insulin. Insulin measurements can lead to the diagnosis or exclusion of insulinoma.
Insulin/glucose ratio.
Plasma C-peptide. insulin secretion in insulin-treated diabetics cannot be assesed by the measurement of plasma insulin since the insulin given therapeutically will also be measured. However, insulin and its associated connecting-peptide (or C-peptide) are secreted by the islet cells in equimolar amounts and thus measurement of C-peptide levels together with insulin can differentiate between hypoglycaemia due to insuloma (high C-peptide ) and that due to exogenous insulin (low C-peptide ).
Specific causes of hypoglycaemia
Over 99% of all episodes of hypoglycaemia occur in insulin-dependent diabetic patients.
Reasons for hypoglycaemia in the diabetics include:
· insufficient carbohydrate intake
· excess of insulin
· strenuous exercise
· excessive alcohol intake
Other causes of hypoglycaemia may be divided in two groups: those which produce hypoglycaemia in the fasting patient, and those in which the low glucose concentration is a response to a stimulus (reactive hypoglycaemia).
Fasting hypoglycaemia
Causes of fasting hypoglycaemia include:
· Insulinoma. ß-cell islet tumors of the pancreas may produce insulin both inappropriately and in excess.
· Cancer. hypoglycaemia is associated with advanced malignancy.
· Hepatic disease.
· Addison’s disease
· Sepsis
Reactive hypoglycaemia
In reactive hypoglycaemia patients may become hypoglycemic in response to:
· Drugs.
· Food: post-prandial hypoglycaemia. Accelerated gastric emptying after gastric surgery (dumping syndrome) may give rise to this condition.
· Alcohol.
Neonatal hypoglycaemia
The diagnosis and treatment of hypoglycaemia in the neonate is particularly important because of the high risk of hypoglycaemic brain damage. There are a number of important causes:
Babies of diabetic mothers. A fetus that is exposed to maternal hyperglycaemia will have pancreatic islet cell hyperplasia and elevated insulin levels. After delivery the neonate is unable to suppress its inappropriately high insulin levels and will develop hypoglycaemia.
Intra-uterine retardation. Small-for-dates babies may have inadequate liver glycogen stores.
Inborn errors of metabolism. Galactosaemia and glycogen storage disease are examples.
Role of the medical laboratory in diabetes mellitus
The laboratory has an essential role in the diagnosis and management of diabetes mellitus.
Routine laboratory indicators for the control of management of diabetes:
· Glucose (blood, urine)
· Ketones (urine)
· OGTT
· HbA1c
· Fructosamine
· Urinary albumin excretion
· Creatinine / urea
· Proteinuria
· Plasma lipid profile
Glucose determination
The simplest indicator of the adequacy of carbohydrate metabolism of a patient is the blood glucose concentration. However glucose is rapidly metabolized in the body. Therefore, the glucose concentration reflects the immediate status of carbohydrate metabolism, and does not allow a retrospective or prospective evaluation of glucose metabolism.
Glucose is measured in different specimens, including
– whole blood (capillary or venous blood)
– haemolysate
– plasma
– serum
– de-proteinized blood
– urine
– CSF
Stability of specimen: Venous blood: at 20°C: decrease of 10-15 %/h at 4°C: decrease of 20 % in 24 h
Stabilizer: NaF (6 g/L) + Maleinimide (0.1 g/L blood)
EDTA (1,2-2 g/L) or EDTA + maleinimide
Plasma/serum: at 20 °C: decrease of 15 % in 24 h
Deproteinized serum: stable over days and weeks
Interferences: Anticoagulants, drugs, glutathione, ascorbic acid, α-methyldopa
Pre-analytical effects: Posture, exercise, food ingestion, smoking, transport/preservation of specimen
Methods for blood glucose determination
Several methods are available for glucose determination. The methods for glucose analysis are the following:
3. Chemical methods:
· ortho-toluidine
· neocuproine
· ferricyanide
4. Enzymatic methods
· hexokinase-G6PDH
· glucose dehydrogenase
· glucose oxidase-peroxidase (ABTS)
· glucose oxidase (GOD) with other indicator reactions
The concentration of glucose in cerebrospinal fluid is about 60 % of the plasma value. If CSF is contaminated with bacteria or additional cells, the glucose concentration may be much lower.
Urine glucose
Urine fractions should be analysed immediately or preserved at pH <5 to inhibit bacterial metabolism of glucose or should be stored at
Advantages:
· rapid
· inexpensive
· non-invasive
· qualitative tests or semi-quantitative tests
UrINE ANALYSIS.
The kidneys are paired organs, which are responsible for the constancy of the internal environment in the organism and elimination of the metabolism end-products.The kidneys regulate water-electrolyte balance, acid-base balance, excretion of metabolic wastes, osmotic pressure. Besides, they take part in the regulation of blood pressure and R.B.C production.
1. Structure and function of the kidneys
Each kidney is composed of 2 layers: the cortex or outer layer is brownish- red and the medulla or inner layer is lighter in colour. The nephron is the functioning unit of the kidney. Each kidney contains more than a million of nephrons.
It consists of renal corpuscle, which contains glomerulus surrounded by hollow capsule (Bowman’s capsule). Besides each nephron contains: proximal convoluted tubules, a descending limb of the loop of Henle, collecting tubules and distal convoluted tubules.
The 2 principal types of nephron are classified according to their position in the kidneys:
1. Cortical nephrons (85%), which are situated in the cortex.
2. Yuxtamedullary nephrons (15%)
The kidneys are the most important organs of excretion. A human dies when the kidneys are not functioning for 4-6 days.
Mechanism of the urine formation
How urine is formed?
There are 3 basic renal processes: filtration, reabsorption and secretion. Glomerular filtration is caused by difference between glomerular pressure (
The effective filtration pressure is approximately 70mmHg -(30mmHg + 20mmHg) =20mmHg . Oncotic + capsular pressure must be lower than glomerular pressure. As a result of the filtration primary urine is formed. Assuming that the kidneys are healthy and filter approximately 20% of the plasma they receive each minute, they will produce 180 to
Some of the blood that passes through the kidneys, in the other words, is “cleared” from waste products.
If a substance is neither reabsorbed nor secreted by the tubules, the amount of excreted per minute in the urine will be equal to the amount that is filtered out of the glomeruli.
The renal plasma clearence is the volume of plasma from which a substance is completely removed in 1 minute by excretion in the urine.
Renal plasma clearence is calculated using formula:
C = V*U
P
Where C-clearence
V-urine formation volume per minute (ml per min)
U-concentration of substance in urine (mg%)
P-concentration of substance in plasma (mg%)
For example: clearence of inulin, creatinine is equal to 125 ml per min (because they are not being reabsorbed); These substances are used for the determination of renal plasma clearence in medicine.
If clearence >125, it means that substance is intensively secreted in tubules.
If clearence <125, it means that there is some inflammatory process of the kidneys (nephritis), which caused azotemia.
Tests of glomerular function
The GFR depends on the net pressure across the glomerular membrane, the physical nature of the membrane and its surface area, which in turn reflects the number of functioning glomeruli. All three factors may be modified by disease, but in the absence of large changes in filtration pressure or in the structure of the glomerular membrane, the GFR provides a useful index of the numbers of functioning glomeruli. It gives an estimate of the degree of renal impairment by disease.
Accurate measurement of the GFR by clearance tests requires determination of the concentrations, in plasma and urine, of a substance that is filtered at the glomerulus, but which is neither reabsorbed nor secreted by the tubules; its concentration in plasma needs to remain constant throughout the period of urine collection. It is convenient if the substance is present endoge-nously, and important for it to be readily measured. Its clearance is given by
Clearance = U- V/P
where U is the concentration in urine, V is the volume of urine produced per minute and P is the concentration in plasma. When performing this calculation manually, care should be taken to ensure consistency of units, especially for the plasma and urine concentrations.
Inulin (a complex plant carbohydrate) meets these criteria, apart from the fact that it is not an endogenous compound, but needs to be administered by IV infusion. This makes it completely impractical for routine clinical use, but it remains the original standard against which other measures of GFR are assessed.
Measurement of creatinine clearance
Creatine is synthesised in the liver, kidneys and pancreas, and is transported to its sites of usage, principally muscle and brain. About 1-2% of the total muscle creatine pool is converted daily to creatinine through the spontaneous, non-enzymatic loss of water. Creatinine is an end product of nitrogen metabolism, and as such undergoes no further metabolism, but is excreted in the urine. Creatinine production reflects the body’s total muscle mass.
Creatinine meets some of the criteria mentioned above. Creatinine in the plasma is filtered freely at the glomerulus, but its concentration may not remain constant over the period of urine collection. A small amount of this filtered creatinine undergoes tubular reabsorption. A larger amount, up to 10% of urinary creatinine, is actively secreted into the urine by the tubules. Its measurement in plasma is subject to analytical overestimation. In practice, the effects of tubular secretion and analytical overestimation tend to cancel each other out at normal levels of GFR, and the creatinine clearance is a fair approximation to the GFR. As the GFR falls, however, creatinine clearance progressively overestimates the true GFR.
Creatinine clearance is usually about 110 mL/min in the 20-40-year-old age group. Thereafter, it falls slowly but progressively to about 70 mL/min in people over
Creatinine clearance or plasma [creatinine]?
Measurement of plasma [creatinine] is more precise than creatinine clearance, as there are two extra sources of imprecision in clearance measurements, that is, timed measurement of urine volume and urine [creatinine]. Accuracy of urine collections is very dependent on patients’ cooperation and the care with which the procedure has been explained or supervised. The combination of these errors causes an imprecision (1 SD) in the creatinine clearance of about 10% under ideal conditions with ‘good’ collectors; this increases to 20-30% under less ideal conditions. This means that large changes in creatinine clearance may not reflect any real change in renal function.
It will be apparent that creatinine clearance measurements are potentially unreliable. Although creatinine clearance measurements are commonly made, accurate measurement of GFR is not often required. Indications for its measurement include determining the dose of a number of potentially toxic drugs that are cleared from the body by renal excretion, investigation of patients with minor abnormalities of renal function and assessment of possible kidney donors.
In most circumstances, however, assessment of glomerular function can be made and changes in GFR over time can be monitored, biochemically, by measurement of plasma [creatininej rather than by measurement of creatinine clearance, because
1 plasma [creatinine] normally remains fairly constant throughout adult life, whereas creatinine clearance declines with advancing age;
2 plasma [creatinine] correlates as well with GFR as does creatinine clearance in patients with renal disease;
3 measurements of plasma [creatinine] are as effective in detecting early renal disease as creati nine clearance, despite the form of the relation ship described above, because of the imprecision in measuring creatinine clearance;
4 sequential plasma [creatinine] measurements enable the progress of renal disease to be followed with better precision than creatinine clearance.
Low plasma [creatinine]
A low [creatinine] is found in subjects with a small total muscle mass (Table 4.1). A low plasma [creatinine] may therefore be found in children, and values are, on average, normally lower in women than in men. Abnormally low values may be found in wasting diseases and starvation, and in patients treated with corticosteroids, due to their protein catabolic effect. Creatinine synthesis is increased in pregnancy, but this is more than offset by the ncombined effects of the retention of fluid and the physiological rise in GFR that occur in pregnancy, so plasma [creatinine] is usually low.
High plasma [creatinine]
Plasma [creatinine] tends to be higher in subjects with a large muscle mass (Table 4.1). Other non-renal causes of increased plasma [creatinine] include the following:
1 A high meat intake can cause a temporary increase.
2 Transient, small increases may occur after vigor ous exercise.
3 Some analytical methods are not specific for creatinine. For example, plasma [creatinine] will be overestimated by some methods in the presence of high concentrations of acetoacetate or cephalosporin antibiotics.
4 Some drugs (e.g. salicylates, cimetidine) compete with creatinine for its tubular transport mechanism, thereby reducing tubular secretion of
creatinine and elevating plasma [creatinine].
If non-renal causes can be excluded, an increased plasma [creatinine] indicates a fall in GFR. The renal causes of this include:
1 any disease in which there is impaired renal perfusion (e.g. reduced blood pressure, fluid depletion, renal artery stenosis);
2 most diseases in which there is loss of functioning nephrons (e.g. acute and chronic glomerulonephritis);
3 diseases where pressure is increased on the tubular side of the nephron (e.g. urinary tract obstruction due to prostatic enlargement).
Other tests of glomerular function
Isotope tests
A number of isotopic markers (e.g. 51Cr-EDTA, 99Tc-DTPA) are almost entirely cleared from the circulation by glomerular filtration. They are injected or infused, and the measurement of their disappearance from the circulation or appearance in urine can be used to calculate the GFR. These tests have largely superceded the use of inulin clearance, but are not widely used in routine clinical practice.
ß2-microglobulin
ß2-microglobulin is a small (11.8 kDa) protein found on the cell surface of all nucleated cells, as part of the class 1 major histocompatibility complex. It is shed into the blood, where it is normally present in low concentrations. Its small size allows it to pass freely through the glomerular membrane, following which it is reabsorbed and catabolised in the proximal tubules. As glomerular filtration falls, the concentration of ß2-microglobulin rises, making it a good indicator of GFR in normal people, since it is not affected by muscle mass or diet. However, its concentration also increases in a number of malignancies and inflammatory conditions. The prognosis in multiple myeloma is adversely influenced by increasing tumour mass and by declining renal function, both of which cause ß2-microglobulin to rise, making it a helpful prognostic indicator in this condition.
Cystatin C
Cystatin C is a cysteine protease inhibitor produced by all nucleated cells. It is a small (13 kDa) basic protein which is freely filtered by the glomerulus and almost completely reabsorbed and catabolised by the proximal tubules. Serum levels of cystatin C are independent of weight, height, muscle mass, age (over 1 year) or sex, and it has a stable production rate. Serum levels correlate well with GFR, performing at least as well as creatinine, and being less subject to confounding influences. However, at present its measurement is much more expensive and not as rapid as the measurement of creatinine, so despite promise as a measurement, it is unlikely to be widely adopted in the near future.
2. Mechanisms of reabsorption in kidneys’ tubules.
The biggest part of primary urine during its transference through kidney tubules (the length of all kidney tubules is more than 100km) return many components into blood. Approximately all important for organism substances are reabsorbed. The mechanisms involved in this process may be divided into 2 categories : simple diffusion and active transport.
The main portion of substances is reabsorbed by active transport which requires the use of metabolic energy. That’s why system of active transport is very developed in kidneys tubules. High activity of Na+/K+ ATPase creates Na+/K+ gradient for secondary active transport of different substances. All the substances are divided into 3 groups due to their extent of reabsorption in proximal tubules :
1.Substances which are actively reabsorbed
2.Substances which are reabsorbed not enough
3.Substances which are not reabsorbed
Ions of sodium, chloride, magnesium, calcium, water, glucose and other monosaccharides, amino acids, phosphates, hydrocarbonates, proteins, etc are actively reabsorbed.
Glucose and proteins are reabsorbed approximately all, amino acids – up to 93%, water – up to 96%, NaCl- up to 70%, the other substances- more than 50%. Reabsorption of Na ions by the tubular epithelial cells is generally regarded as an active transport. Firstly Na ions pass from the kidney tubules into the epithelial cells and from there- into extracellular space.
Tubular reabsorption of Cl and HCO3– occurs passively in association with reabsorption of Na+. Water is absorbed isoosmotically with Na and also by flowing along the osmotic gradient due to increase of osmotic pressure in extracellular space. From there substances pass into capillaries.
Glucose and amino acids are transported by the special mediators in association with Na. They use energy of Na+ – gradient on membrane Ca and Mg are reabsorbed by the help of special ATPase. Protein is reabsorbed by endocytosis.
Urea and uric acid belong to substances which are being reabsorbed not enough. They are transported by simple diffusion into extracellular space, and from there-in loop of Henle.
Creatinine, mannitol, inulin – are substances which are not being reabsorbed.
Functional significance of different parts of kidney tubules in the urine formation is heterogeneous. Descending and ascending limbs of the loop of Henle form the countercurrent system which takes part in concentration and dilutation of the urine due to the normal range for the specific gravity of urine which is from 1.002 to 1.030.
Liquid, which is transferred from the proximal tubule to descending limb of the loop of Henle, passes in kidney zone where concentration of osmoactive substances is higher, than in cortex. The walls of the ascending limb of the loop of Henle are not permeable to water. Salt (NaCl) is extruted into the surrounding tissue fluid. The descending limb does not actively transport salt. It is however, permeable to water.
Since the surrounding interstitial fluid is hypertonic to the filtrate in the descending limb, water is drawn out of the descending limb by osmosis and enters blood capillaries. This system results in a gradually increasing concentration of renal tissue fluid from the cortex to the inner medulla; the osmolality of tissue fluid increases from 300mOsm/l to 1450mOsm/l.
Tests of tubular function
Specific disorders affecting the renal tubules may affect the ability to concentrate urine or to excrete an appropriately acidic urine, or may cause impaired reabsorption of amino acids, or glucose, or phosphate, etc. In some conditions, these defects occur singly; in others, multiple defects are present. Renal tubular disorders may be congenital or acquired, the congenital disorders all being very rare. Chemical investigations are needed for specific identification of these abnormalities and may include amino acid chromatography, or investigation of calcium and phosphate metabolism, or an oral glucose tolerance test (OGTT). The functions tested most often are renal concentrating power and the ability to produce an acid urine.
The healthy kidney has a considerable reserve capacity for reabsorbing water, and for excreting H+ and other ions, only exceeded under exceptional physiological loads. Moderate impairment of renal function may reduce this reserve, and this is revealed when loading tests are used to stress the kidney. Tubular function tests are only used when there is reason to suspect that a specific abnormality is present.
Fluid deprivation test
This test is effectively a bioassay of vasopressin, which is itself difficult to measure. The test can be hazardous in a patient excreting large volumes of dilute urine, and requires close supervision. There are a number of ways of performing a fluid deprivation test, differing in detail but all involving fluid deprivation over several hours, ensuring that the patient under observation takes no fluid, and that excessive fluid losses do not occur. Local directions for test performance should be followed. For instance, beginning at 10 pm, the patient is told not to drink overnight, and urine specimens are collected while the patient continues not to drink between 8 am and 3 pm the next day. During the test, the patient should be weighed every 2 h, and the test should be stopped if weight loss of 3-5% of total body weight occurs. Blood and urine specimens are collected for measurement of osmolality. Normally, there is no increase in plasma osmolality (reference range 285-295 mmol/kg) over the period of water deprivation, whereas urine osmolality rises to 800 mmol/kg or more. A rising plasma osmolality and a failure to concentrate urine are consistent with either a failure to secrete vasopressin or a failure to respond to vasopressin at the level of the distal nephron. When this pattern of results is obtained, it is usual to proceed immediately to perform the DDAVP test.
DDAVP test
The patient is allowed to drink a moderate amount of water at the end of the fluid deprivation test, to alleviate thirst. An intramuscular injection of DDAVP is then given, and urine specimens are collected at hourly intervals for a further 3 h and their osmolality measured.
Interpretation of tests of renal concentrating ability
These tests are of most value in distinguishing among hypothalamic-pituitary, psychogenic and renal causes of polyuria (Table 4.3).
Patients with diabetes insipidus of hypothalamic-pituitary origin produce insufficient vasopressin; they should therefore not respond to fluid deprivation, but should respond to the DDAVP. As a rule, these patients show an increase in plasma osmolality during the fluid deprivation test, to more than 300 mmol/kg, and a low urine osmolality (200-400 mmol’Kg). There is a marked increase in urine osmolality, to 600 mmol/kg or more, in the DDAVP test.
Patients with psychogenic diabetes insipidus should respond to both fluid deprivation and DDAVP. In practice, however, renal medullary hypo-osmolality often prevents the urine osmolality from reaching 800 mmol/kg after fluid deprivation or DDAVP injection in these tests, as is normally performed. Also, the chronic suppression of the physiological mechanism that controls vasopressin release may impair the normal hypothala-mic response to dehydration. These patients have a plasma osmolality that is initially low, but which rises during the tests. However, fluid deprivation may have to be continued for more than 24 h in these patients before medullary hyperosmolality is restored; only then do they show normal responses to fluid deprivation or to DDAVP injection.
Polyuria of renal origin may be due to inability of the renal tubule to respond to vasopressin, as in nephrogenic diabetes insipidus. In this condition, there is failure to produce a concentrated urine in response either to fluid deprivation or to DDAVP injection, the urinary osmolality usually remaining below 400 mmol/kg; in these patients, plasma osmolality increases as a result of fluid deprivation.
Fanconi’s syndrome
Fanconi’s syndrome may be inherited (e.g. in cystinosis) or secondary to a number of other disorders (e.g. heavy metal poisoning, multiple myeloma). The syndrome comprises multiple defects of proximal tubular function. There are excessive urinary losses of amino acids (generalised amino aciduria), phosphate, glucose and sometimes HCO3, which gives rise to a proximal renal tubular acidosis. Distal tubular functions may also be affected. Sometimes globulins of low molecular mass may be detectable in urine, in addition to the amino aciduria
RENAL REGULATION OF ACID-BASE BALANCE
The kidneys help to regulate the blood pH, together with respiratory system and the blood buffer systems. Blood buffer systems very quickly react to violation of pH (in 0.5-1 min); lungs influence on hydrogen ions concentration in 1-3 min ; and kidney is the latest regulator of pH (in 10-20 hours). There are 2 main mechanisms which are responsible for the kidneys regulation of blood pH: reabsorption of sodium and secretion of hydrogen ions.
1) Reabsorption of sodium ions during transformation the alkaline phosphate Na2HPO4 of the blood to the acidic phosphate (NaHPO4) which is eliminated in the urine.
2) When the urine is acidic, HCO3– combines with H+ to form carbonic acid. Carbonic acid in the filterate is then converted to CO2 and H2O by the action of carbonic anhydrase. Carbonic acid dissociates to HCO3– and H+. Then H+ (acid) excreted in the urine and HCO3–(base) passes in to the blood as NaHCO3 and decreases the acidity.
3) Ammonia (NH3) is a base that is formed from the amino acid glutamine within the tubular cells. It crosses into tubular lumen to combine with H+ to form ammonium (NH4). This effectively prevents accumulation of H+ ions in the fluid, and therefore permits continued exchange of H+ for Na+ ions. The amount of Na+ ions abbsorbed in the distal tubule is consequently reflected in the amount of both H+ and NH4+ ions in the urine.
8. Properties and urine’s composition
The amount of urine (diuresis) excreated by a healthy man is 1000-2000 ml per 24 hours. Daily amount of urine, which is lower than 500 ml and higher than 2000 ml, of adults is considered to be pathological. Men’s diuresis is a little bit higher than women’s one, and it is 1500-2000 ml, and women’s diuresis is 1000-1600 ml. Twenty four hour’s diuresis can change depending on the kind of a diet, conditions of work, the temperature of the environment and ets.
Drinking a lot of water causes the increase of diuresis to 2000-3000 ml, and decrease of water drinking causes the decrease of diuresis to 700 ml and even less. Consuming of fruits, berries and vegetables, rich in water also increase diuresis, but dry products, especially salted, lower it. The volume of urine is also lowered during a work in hot shops when a man loses water mostly through sweating.
Diuresis’s increase (poliuria) is observed with many diseases and while using different diuretics. A lot of urine is excreted by the patient who are ill with diabetes mellitus and diabetes insipidus.
Twenty four hour’s decrease of urine excretion (oliguria) is observed while having fever, diarhea, nausea, acute nephritis, heart deficieny and in some other cases.
When a man is lead or arsenic poisoned, is upset, has nephritis, the full stop of urine excretion (anuria) is observed. Prolonged anuria causes uremia. According to standard, urine is discharged 3-4 times more by day – light time than at night. But in some pathological conditions (the beginning of heart decompensation, diabetes, nephritis) become apparent by predominance of night discharge compare to day time. Such condition is called nicturia.
Urine’s colour. Usually urine is straw -yellow. It’s main pigment is urochrome which is formed from urobilin or urobilinogen during their interaction with some peptides. Some other pigments influence on the urine’s colour, that’s uroerytryn which is obviously derivate of melanin, uroporphyrins, riboflavin and others. During the conservation obviously as a result of urobilinogen oxidation, urine darkens. Such urine is observed during bilirubin’s excretion when a man is ill with obstructive or hepatic jaundice.
Concentrated urine, which is excreted in large quantities and has high specific gravity, is of bright-yellow color.
Pale urine has low specific gravity and is excreted in large quantities.
Urine can become of different colour shades when a patient has pathological changes. Urine is red or pink-red when a patient is ill with hematuria, hemoglobinuria, when he takes amidopirin, santonin and other medicines. High concentration of urobilin and bilirubin can cause dark-red colour of urine.Green or blue colour of urine is observed while albumin is rotting in the bowels and as a result, indoxylsulphuric acids are produced. The last ones while decomposing produce indigo.
Transparency. Fresh urine is transparent. Not fresh urine opacificates because of mucins and the epithelium of the mucosal membrane of urethras. Urine’s opacification is caused also by the crystals of oxalic acid (oxalates) and uric acid (urates). During durable urine standing mostly urates are in fall-our, which, adsorpting pigments, cause its opacification. Calcium and magnesium phosphates are in fall-out in urine with alkaline reaction. Alkaline character of urine which is falling out is caused by the decomposition of urea under the influence of urine’s microflora to ammonia. Ammonia makes urine alkaline that causes the fall-out of mentioned solts and urine’s darkening.
Urine also becomes turbid when a patient is ill with inflamatory process of urethra ducts while pus, proteins, blood cells falling into urine.
For the diagnostics of some diseases urine is acidified and warmed up. If after this process cloudiness disappears it means that it is caused by calcium or magnesium phosphates or urates. If cloudiness doesn’t disappear it means that it is caused by pus, epitheliym cells and by other admictures.
Urine’s specific gravity depends on the concentration of dissolving substances. During twenty four hours urine’s density changes from 1.002 to
Increase of the density during a normal diuresis or poliuria is observed with that patients who discharge a great amount of organic and nonorganic substanses. Urine of the person with diabetes mellitus contains sugar, ketone bodies and other substances, which cause not only poliuria, but a high density ( to 1.035). Daily diureses with low specific density of urine is observed among the patients with diabetes insipidus. Urine with low density which is similar to primary urine (1.010) is constantly discharged when a person has a complicated form of renal failure. Such condition is called sthenuria, and it speaks about the disturbance of the concentrational functions of kidneys.
Low density of urine which have patients with diabetes incipidus (1.001-1.004) is the result of the disturbance of reverseble reabsorption of water in kidney’s canaliculi becouse of lack of antidiuretic hormone.
Oliguria which accompanies acute nephritis is characterized with high urine’s density
Urine’s reaction. Normally, having mixed food urine is acidic or light acidic (pH=5.3-6.8). Urine with pH=6 is usually taken as the norm. Eating mostly meat food and proteins gives urine acidic reaction, while eating vegetables it become alkaline. Acidic reaction of urine is mainly caused by onesubsubstituted phosphaties, mostly NaH2PO4 and KH2PO4. Twosubstituted phosphaties or biocarbonate potassium or sodium predominate in alkaline urine. Considerable emimence of alkaline substances in blood is accompanied with biocarbonates excretion with urine that raises pH from 6.0 to 7,5-7.7.
Alkaline reaction of urine is observed in patients who are ill with the cystitis (inflammation of urinary bladder) which is connected with urea decomposition and ammonia formation.
The same reaction is observed after vomiting, drinking of alkaline mineral waters and so on.
Clearly acidic reaction is notable for patients who are ill with diabetes mellitus, during fever and starvation.
Urine’s smell. Fresh urine has a specific smell mainly caused by volatile acids which are available in it. Urine which is preserved, is influenced by microorganisms, specifically by the decomposition of urea with ammonia forming. The last one causes acute ammonia smell. Healthy people’s urine can have different smell, depending on kind of meals. Having some garlic, horseradish, onion gives urine specific smell. Taking medicines and also some diseases can give urine specific smell to.
8.1 Chemical composition of the urine
There are a lot of different organic and non- organic substances in the urine (about 200 ).
They are metabolism end- products in the kidneys and other organs and tissues of the organism.
8.2 Organic components of the urine
Proteins. Healthy man excretes about 30 mg of proteins with urine per day. This quantity of the protein is not determined by ordinary lab. methods. As a rule low molecular proteins are eliminated , such as enzymes( pepsin, trypsin , amilase, ets.) , albumins. The increasing of protein level in urine is called proteinuria. There are 2 kinds of proteinuria: renal (real) and extrarenal (unreal or false).
Renal proteinuria is caused by organic demage of nephrons , due to blood proteins (albumins and globulins) occur in urine. For example inflammation of glomeruluses (glomerulonephritis )or nephrosis (violations of proteins reabsorption in tubules).
Extrarenal proteinuria- availability of proteins in urine due to diseases of urinary tract. (inflammation of urinary bladder, urethritis). Patients with such diseases may loose 20
Urea is the main end- product of the catabolism of amino acids and is the substance in which is incorporated , for purposes of excretion, the bulk of the nitrogen provided to the organism in excess of its needs. Nitrogen of urea is equal to 80 -90 %of total nitrogen in urine.
An adult eliminates 20-
The increasing of urea concentration:
1. Excess of proteins in the diet
2. Diabetes mellitus
3. Cancer
4. Fever
The decreasing of urea concentration:
1. Lack of proteins in the diet
2. Liver diseases
3. Acidosis
4. Intensive growth of the organism
Pre-renal uraemia may develop whenever there is impaired renal perfusion, and is essentially the result of a physiological response to hypovolaemia or a drop in blood pressure. This causes renal vaso-constriction and a redistribution of blood such that there is a decrease in GFR, but preservation of tubular function. Stimulation of vasopressin secretion and of the renin-angiotensin-aldosterone system causes the excretion of small volumes of concentrated urine with a low Na content. This reduced urine flow in turn causes increased passive tubular reabsorption of urea. Thus shock, due to burns, haemorrhage or loss of water and electrolytes (e.g. severe diarrhoea) may lead to increased plasma [urea]. Renal blood flow also falls in congestive cardiac failure, and may be further reduced if such patients are treated with potent diuretics. If pre-renal uraemia is not treated adequately and promptly by restoring renal perfusion, it can progress to intrinsic renal failure.
Increased production of urea in the liver occurs on high protein diets, or as a result of increased protein catabolism (e.g. due to trauma, major surgery, extreme starvation). It may also occur after haemorrhage into the upper GI tract, which gives rise to a ‘protein meal’ of blood.
Plasma [urea] increases relatively more than plasma [creatinine] in pre-renal uraemia. This is because tubular reabsorption of urea is increased significantly in these patients, whereas relatively little reabsorption of creatinine occurs.
Renal uraemia may be due to acute or chronic renal failure, with reduction in glomerular filtration. Plasma [urea] increases until a new steady state is reached at which urea production equals the amount excreted in the urine, or continues to rise in the face of near-total renal failure. Although frequently measured as a test of renal function, it is always important to remember that plasma [urea] may be increased for reasons other than intrinsic renal disease (pre-renal and post-renal uraemia).
Post-renal uraemia occurs due to outflow obstruction, which may occur at different levels (i.e. in the ureter, bladder or urethra), due to various causes (e.g. renal stones, prostatism, genitourinary cancer). Back-pressure on the renal tubules enhances
back-diffusion of urea, so that plasma [urea] rises disproportionately more than plasma [creatinine]. Impaired renal perfusion and urinary tract obstruction, each in itself possible causes of uraemia, may in turn cause damage to the kidney and thus renal uraemia.
Uric acid:
An adult eliminates 0.6 – 1g of uric acid with urine per day.
The increasing of uric acid concentration:
1. Feeding products, which contain many nucleoproteins(meat , fish eggs etc)
2. Leucosis, burns
3. Some Drugs (Aspirin)
4. Violations of proteins metabolism (gout)
The decreasing of uric acid concentration:
1. Diet poor in proteins and rich in carbohydrates
Intermediate products of purine metabolism are also excreated with urine (xanthine, hypoxanthine – 20-50 mg per day).
Creatinine and Creatine:
An adult excretes of 1-
Synthesis of creatine, from which creatinine is formed, is in the kidneys and liver. The decreasing concentration:
1. The kidneys and the liver lesion
2. Violations of protein metabolism
3. Atrophy of muscle
Creatinine is neither reabsorbed nor secreted by the tubules, the amount excreted per minute in the urine will be equal to the amount that is filtered out of the glomeruli.
Creatinine is used for the determination of renal plasma clearance.
The increasing of the creatinine concentration:
–Some infections
-Intoxications
The decreasing of creatinine concentration:
– Violations of filteration in kidneys. Children excrete more creatine than adults, females- more, than males.
Cretinuria takes place in old people due to muscle atrophia .
Amino Acids:
An adult excretes about 2-
1. Splitting of the tissue proteins
2. Violations of liver functions
There are some genetic defects in the metabolism of seperate amino acid. For example:
1) Phenylketonuria: Which is caused by enzyme phenylalanine-4-monoxygenase absence. In this case a pathway of phenylalanine breakdown and tyrosine is not formed. To determine phenylketonuria is used FeCl3 ( fresh urine +2-3 drops of FeCl3 solution and in 2-3 min observe appearance of dark-green colour).
2) Alkaptonuria: The urine of people genetically defective in homogentisic acid 1,2-dioxygenase contains homogentisic acid, which when made alkaline and exposed to oxygen, turns dark because it is oxidised and polymerized. to a black melanine pigment.
Paired compounds:
Hippuric acid (benzoglycine) is formed by the conjugation in peptide linkage of benzoic acid and glycine. In a man this occours largely in the liver and also in the kidneys. Benzoglycine is excreted in the urine in amounts ranging from 0.6 to
Indican : It is excreted in the urine in amounts ranging from 10-25 mg daily.
The increasing of indican concentration :
1. Intestinal obstruction
2 Generalized peritonitis.
3. Decomposition of tissue protein, for example, tuberculosis
Organic compounds:
In the urine of healthy man some organic acids are usually observed (for example, acetoacetate ). Some lipids (cholesterol) are present in urine in small amounts.
Vitamins:
Allmost all vitamins are excreted with urine. Most of all water-soluble vitamins such as thiamine: 0.1-0.3mg, riboflavin: 0.5-0.8mg ,ascorbic acid: 20-30mg . In medicine wide-spread is a method of determining quantity mg. of vitamin C,which is excreted in urine per hour.In a person 1mg of vitamin C is excreted per hour.
Hormones:
Some hormones are present in urine. Androgenic compounds of 17- ketosteroids structure are found in the urine of normal person in amounts ranging from 15-25 mg. The increasing of this quantity may be caused by adrenocortical tumours.
Urobilin (stercobilin): always is present in small amounts in urine. It’s concentration increases when liver looses property to decompose urobilinogen from intestine(haemolytic jaundice and hepatic jaundice).
Bilirubin:
Urine of healthy individual contains a small amount of bilirubin, which is not determined by ordinary lab. methods.
Causes of bilirubinuria:
1. Obstruction of bile canaliculi and bile duct.
2. Damage of liver cells.
Urine will have special colour like dark beer, then it becomes yellow-green, due to oxidation of bilirubin into biliverdin.
Glucose:
Urine of healthy person contains small amounts of glucose, which is not determine by urinary lab methods.
Glucose is normally completely reabsorbed in the proximal tubule. But in the patient with diabetes mellitus , content of glucose in urine may be 5-10%.
Galactose:
Galactose is metabolised mainly by liver. Alimentary galactosuria, which is related to the ingestion of milk and milk products.
Iew borns galactosuria very often combines with lactosuria.
Galactose tolerance:
After ingestion of 40 mg. of galactose , quantity of galactose is detected in urine per every hour.
Iormal conditions galactose is excreted in urine in first 2 hours.
Fructose:
Fructose may appear in the urine under the following circumstances;
1. Alimentary fructosuria(fruits, berries, honey).
2. Unsatisfactory hepatic function.
3. Diabetes mellitus.
Pentose:
Pentose may appear in the urine under the following circumstances;
1. Alimentary pentosuria, occuring iormal individuals after the ingestion of large quantities of fruts which have high quantity of pentose content (cherries, grapes, plums).
2.Essential Pentosuria.
It is genetically determined, has a familial incidence.
Ketone bodies:
Iormal conditions daily urine content is 20-50mg of ketone bodies. It is not determined by ordinary lab. methods.
Ketonuria may occur in a variety of clinical conditions;
1. Diabetes mellitus (20-50mg per day).
2. Carbohydrate starvation.
3. Thyrotoxicosis and fever.
Blood:
When red blood cells appear in urine ( hematuria), it means that there are some damage of the kidney or urinary tract. Hemoglobinuria — presence of free hemoglobin in urine , is a result of hemolysis (renal infarction, poisons).
Porphyrins:
These are red pigments with a pyrolle structure , which are important components of hemoglobin ,, myoglobin, cytochrome and catalysts.Iormal conditions daily urine contain very small amount of poryphyrin type I (300 mkg).
There are 3 isomeric etioporyphyrins , designated typeI, II, III.
Porphyrinuria may occur in a variety of clinical conditions :
– some liver diseases
– intoxication
– intestine bleeding
– pernicious anemia
8.3 Mineral components of the urine
Iormal conditions daily urine contains 15 to
Sodium chloride is the most wide-spread non-organic substance in urine. It is excreted in amounts ranging from 8 to
About
Iormal conditions daily urine contains 2 to
Calcium and magnesium: In normal conditions daily urine contains 0,1-
Iormal conditions daily urine contains 0,03-
Such low Ca and Mg concentration is because their salts are poorly soluble in water.
Iron:
Iormal conditions daily urine contains about 1 mg of iron.
Excessive breakdown of erythrocytes in hemolytic types of anemia causes the increasing iron concentration.
Phosporus:
Phosphorus is excreted in urine as KH2PO4 or NaH2PO4.
Quantity of excreted phosphate depends on blood pH:
1. Acidosis: Alkaline phophate (NaH2PO4) react with acids and are transformed into acid phosphates(NaHPO4) which are eliminated in the urine.
2. Alkalosis: Acidic phosphates (NaHPO4) react with bases and are transformed into alkaline phosphates (Na2HPO4 ) which are eliminated in the urine.
Ammonia:
The ammonia which is present in the urine is formed in the kidneys from amino acids , such as glutamine and asparagine, for purpose of neutralization of excreted acid.
Quantity of ammonia salts is equal to 3-6% of total urinary nitrogen. Urinary ammonia is increased in many conditions associated with acidosis( diabetes mellitus, starvation , dehydration, etc.)
