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CHOLERA is a specific infectious disease that affects the lower portion of the intestine and is characterized by violent purging, vomiting, muscular cramp, suppression of urine and rapid collapse. It can a terrifying disease with massive diarrhea. The patient’s fluid losses are enormous every day with severe rapid dehydration, death comes within hours.

1. Hyperthyroidism: Elevation of both T4 and T3 values along with decrease of TSH are indicative of primary hyperthyroidism.
2. Increased thyroxine-binding globulin: If concentration of TBG increases, free hormone level falls, release of TSH from pituitary is stimulated, and free hormone concentration is restored to normal. Reverse occurs if concentration of binding proteins falls. In either case, level of free hormones remains normal, while concentration of total hormone is altered. Therefore, estimation of only total T4 concentration can cause misinterpretation of results in situations that alter concentration of TBG.
3. Factitious hyperthyroidism
4. Pituitary TSH-secreting tumor.

1. Primary hypothyroidism: The combination of decreased T4 and elevated TSH are indicative of primary hypothyroidism.
2. Secondary or pituitary hypothyroidism
3. Tertiary or hypothalamic hypothyroidism
4. Hypoproteinaemia, e.g. nephrotic syndrome
5. Drugs: oestrogen, danazol
6. Severe non-thyroidal illness.

1. Diagnosis of T3 thyrotoxicosis: Hyperthyroidism with low TSH and elevated T3, and normal T4/FT4 is termed T3 thyrotoxicosis.
2. Early diagnosis of hyperthyroidism: In early stage of hyperthyroidism, total T4 and free T4 levels are normal, but T3 is elevated.

1. Confirmation of diagnosis of secondary hypothyroidism
2. Evaluation of suspected hypothalamic disease
3. Suspected hyperthyroidism

• A baseline blood sample is collected for estimation of basal serum TSH level.
• TRH is injected intravenously (200 or 500 μg) followed by measurement of serum TSH at 20 and 60 minutes.

1. Normal response: A rise of TSH > 2 mU/L at 20 minutes, and a small decline at 60 minutes.
2. Exaggerated response: A further significant rise in already elevated TSH level at 20 minutes followed by a slight decrease at 60 minutes; occurs in primary hypothyroidism.
3. Flat response: There is no response; occurs in secondary (pituitary) hypothyroidism.
4. Delayed response: TSH is higher at 60 minutes as compared to its level at 20 minutes; seen in tertiary (hypothalamic) hypothyroidism.

• Hyperthyroidism due to Graves’ disease, toxic multinodular goiter, toxic adenoma, TSH-secreting tumor.

• Hyperthyroidism due to administration of thyroid hormone, factitious hyperthyroidism, subacute thyroiditis.

• Differential diagnosis of high RAIU thyrotoxicosis:
  – Graves’ disease: Uniform or diffuse increase in uptake
  – Toxic multinodular goiter: Multiple discrete areas of increased uptake
  – Adenoma: Single area of increased uptake
• Evaluation of a solitary thyroid nodule:
  – ‘Hot’ nodule: Hyperfunctioning
  – ‘Cold’ nodule: Non-functioning; about 20% cases are malignant.

Among the endocrine disorders, disorders of the thyroid are common and are only next in frequency to diabetes mellitus. They are more common in women than in men. Functional thyroid disorders can be divided into two types depending on the activity of the thyroid gland: hypothyroidism (low thyroid hormones), and hyperthyroidism (excess thyroid hormones).

1. Regular cycles, mastalgia, and laparoscopic direct visualization of corpus luteum indicate ovulatory cycles. Anovulatory cycles are clinically characterized by amenorrhea, oligomenorrhea, or irregular menstruation. However, apparently regular cycles may be associated with anovulation.
2. Endometrial biopsy: Endometrial biopsy is done during premenstrual period (21st-23rd day of the cycle). The secretory endometrium during the later half of the cycle is an evidence of ovulation.
3. Ultrasonography (USG): Serial ultrasonography is done from 10th day of the cycle and the size of the dominant follicle is measured. Size >18 mm is indicative of imminent ovulation. Collapse of the follicle with presence of few ml of fluid in the pouch of Douglas is suggestive of ovulation. USG also is helpful for treatment (i.e. timing of coitus or of intrauterine insemination) and diagnosis of luteinized unruptured follicle (absence of collapse of dominant follicle). Transvaginal USG is more sensitive than abdominal USG.
4. Basal body temperature (BBT): Patient takes her oral temperature at the same time every morning before arising. BBT falls by about 0.5°F at the time of ovulation. During the second (progestational) half of the cycle, temperature is slightly raised above the preovulatory level (rise of 0.5° to 1°F). This is due to the slight pyrogenic action of progesterone and is therefore presumptive evidence of functional corpus luteum.
5. Cervical mucus study:
   • Fern test: During estrogenic phase, a characteristic pattern of fern formation is seen when cervical mucus is spread on a glass slide (Figure 862.4). This ferning disappears after the 21st day of the cycle. If previously observed, its disappearance is presumptive evidence of corpus luteum activity.
   • Spinnbarkeit test: Cervical mucus is elastic and withstands stretching upto a distance of over 10 cm. This phenomenon is called Spinnbarkeit or the thread test for the estrogen activity. During the secretory phase, viscosity of the cervical mucus increases and it gets fractured when stretched. This change in cervical mucus is evidence of ovulation.
6. Vaginal cytology: Karyopyknotic index (KI) is high during estrogenic phase, while it becomes low in secretory phase. This refers to percentage of super-ficial squamous cells with pyknotic nuclei to all mature squamous cells in a lateral vaginal wall smear. Usually minimum of 300 cells are evaluated. The peak KI usually corresponds with time of ovulation and may reach upto 50 to 85.
7. Estimation of progesterone in mid-luteal phase (day 21 or 7 days before expected menstruation): Progesterone level > 10 nmol/L is a reliable evidence of ovulation if cycles are regular (Figure 862.5). A mistimed sample is a common cause of abnormal result.

1. Measurement of LH, FSH, and estradiol during days 2 to 6: All values are low in hypogonadotropic hypogonadism (hypothalamic or pituitary failure).
2. Measurement of TSH, prolactin, and testosterone if cycles are irregular or absent:
   Increased TSH: Hypothyroidism
   Increased prolactin: Pituitary adenoma
   Increased testosterone: Polycystic ovarian disease (PCOD), congenital adrenal hyperplasia (To differentiate PCOD from congenital adrenal hyperplasia, ultrasound and estimation of dihydroepiandrosterone or DHEA are done).
3. Transvaginal ultrasonography: This is done for detection of PCOD.

1. Infectious disease: These tests include endometrial biopsy for tuberculosis and test for chlamydial IgG antibodies for tubal factor in infertility.
2. Hysterosalpingography (HSG): HSG is a radiological contrast study for investigation of the shape of the uterine cavity and for blockage of fallopian tubes (Figure 862.6). A catheter is introduced into the cervical canal and a radiocontrast dye is injected into the uterine cavity. A real time X-ray imaging is carried out to observe the flow of the dye into the uterine cavity, tubes, and spillage into the uterine cavity.
3. Hysterosalpingo-contrast sonography: A catheter is introduced into the cervical canal and an echocontrast fluid is introduced into the uterine cavity. Shape of the uterine cavity, filling of fallopian tubes, and spillage of contrast fluid are noted. In addition, ultrasound scan of the pelvis provides information about any fibroids or polycystic ovarian disease.
4. Laparoscopy and dye hydrotubation test with hysteroscopy: In this test, a cannula is inserted into the cervix and methylene blue dye is introduced into the uterine cavity. If tubes are patent, spillage of the dye is observed from the ends of both tubes. This technique also allows visualization of pelvic organs, endometriosis, and pelvic adhesions. If required, endometriosis and tubal blockage can be treated during the procedure.

1. History: This includes type of lifestyle (heavy smoking, alcoholism), sexual practice, erectile dysfunction, ejaculation, sexually transmitted diseases, surgery in genital area, drugs, and any systemic illness.
2. Physical examination: Examination of reproductive system should includes testicular size, undescended testes, hypospadias, scrotal abnormalities (like varicocele), body hair, and facial hair. Varicocele can occur bilaterally and is the most common surgically removable abnormality causing male infertility.
3. Semen analysis: See article Semen Analysis. Evaluation of azoospermia is shown in Figure 861.3. Evaluation of low semen volume is shown in Figure 861.4.
4. Chromosomal analysis: This can reveal Klinefelter’s syndrome (e.g. XXY karyotype) (Figure 861.5), deletion in Y chromosome, and autosomal Robertsonian translocation. It is necessary to screen for cystic fibrosis carrier state if bilateral congenital absence of vas deferens is present.
5. Hormonal studies: This includes measurement of FSH, LH, and testosterone to detect hormonal abnormalities causing testicular failure (Table 861.2).
6. Testicular biopsy: Testicular biopsy is indicated when differentiation between obstructive and non-obstructive azoospermia is not evident (i.e. normal FSH and normal testicular volume).

Hollander’s test (Insulin hypoglycemia test):

In the past, this test was used for confirmation of completeness of vagotomy (done for duodenal ulcer). Hypoglycemia is a potent stimulus for gastric acid secretion and is mediated by vagus nerve. This response is abolished by vagotomy.

In this test, after determining BAO, insulin is administered intravenously (0.15-0.2 units/kg) and acid output is estimated every 15 minutes for 2 hours (8 post-stimulation samples). Vagotomy is considered as complete if, after insulin-induced hypoglycemia (blood glucose < 45 mg/dl), no acid output is observed within 45 minutres.

The test gives reliable results only if blood glucose level falls below 50 mg/dl at some time following insulin injection. It is best carried out after 3-6 months of vagotomy.

The test is no longer recommended because of the risk associated with hypoglycemia. Myocardial infarction, shock, and death have also been reported.

Fractional test meal:

In the past, test meals (e.g. oat meal gruel, alcohol) were administered orally to stimulate gastric secretion and determine MAO or PAO. Currently, parenteral pentagastrin is the gastric stimulant of choice.

Tubeless gastric analysis:

This is an indirect and rapid method for determining output of free hydrochloric acid in gastric juice. In this test, a cationexchange resin tagged to a dye (azure A) is orally administered. In the stomach, the dye is displaced from the resin by the free hydrogen ions of the hydrochloric acid. The displaced azure A is absorbed in the small intestine, enters the bloodstream, and is excreted in urine. Urinary concentration of the dye is measured photometrically or by visual comparison with known color standards. The quantity of the dye excreted is proportional to the gastric acid output. However, if kidney or liver function is impaired, false results may be obtained. The test is no longer in use.

Spot check of gastric pH:

According to some investigators, spot determination of pH of fasting gastric juice (obtained by nasogastric intubation) can detect the presence of hypochlorhydria (if pH>5.0 in men or >7.0 in women).

Congo red test during esophagogastroduodenoscopy:

This test is done to determine the completeness of vagotomy. Congo red dye is sprayed into the stomach during esophagogastroduodenoscopy; if it turns red, it indicates presence of functional parietal cells in stomach with capacity of producing acid.

REFERENCE RANGES

• Volume of gastric juice: 20-100 ml
• Appearance: Clear
• pH: 1.5 to 3.5
• Basal acid output: Up to 5 mEq/hour
• Peak acid output: 1 to 20 mEq/hour
• Ratio of basal acid output to peak acid output: <0.20 or < 20%

• Gastric intubation for gastric analysis is contraindicated in esophageal stricture or varices, active nasopharyngeal disease, diverticula, malignancy, recent history of severe gastric hemorrhage, hypertension, aortic aneurysm, cardiac arrhythmias, congestive cardiac failure, or non-cooperative patient.
• Pyloric stenosis: Obstruction of gastric outlet can elevate gastric acid output due to raised gastrin (following antral distension).
• Pentagastrin stimulation is contraindicated in cases with allergy to pentagastrin, and recent severe gastric hemorrhge due to peptic ulcer disease.

• It is an invasive and cumbersome technique that is traumatic and unpleasant for the patient.
• Information obtained is not diagnostic in itself.
• Availability of better tests for diagnosis such as endoscopy and radiology (for suspected peptic ulcer or malignancy); serum gastrin estimation (for ZE syndrome); vitamin assays, Schilling test, and antiparietal cell antibodies (for pernicious anemia); and tests for Helicobacter pylori infection (in duodenal or gastric ulcer).
• Availability of better medical line of treatment that obviates need for surgery in many patients.

1. To determine the cause of recurrent peptic ulcer disease:
   • To detect Zollinger-Ellison (ZE) syndrome: ZE syndrome is a rare disorder in which multiple mucosal ulcers develop in the stomach, duodenum, and upper jejunum due to gross hypersecretion of acid in the stomach. The cause of excess secretion of acid is a gastrin-producing tumor of pancreas. Gastric analysis is done to detect markedly increased basal and pentagastrinstimulated gastric acid output for diagnosis of ZE syndrome (and also to determine response to acidsuppressant therapy). However, a more sensitive and specific test for diagnosis of ZE syndrome is measurement of serum gastrin (fasting and secretin-stimulated).
   • To decide about completeness of vagotomy following surgery for peptic ulcer disease: See Hollander’s test.
2. To determine the cause of raised fasting serum gastrin level: Hypergastrinemia can occur in achlorhydria, Zollinger-Ellison syndrome, and antral G cell hyperplasia.
3. To support the diagnosis of pernicious anemia (PA): Pernicious anemia is caused by defective absorption of vitamin B₁₂ due to failure of synthesis of intrinsic factor secondary to gastric mucosal atrophy. There is also absence of hydrochloric acid in the gastric juice (achlorhydria). Gastric analysis is done for demonstration of achlorhydria if facilities for vitamin assays and Schilling’s test are not available (Achlorhydria by itself is insufficient for diagnosis of PA).
4. To distinguish between benign and malignant ulcer: Hypersecretion of acid is a feature of duodenal peptic ulcer, while failure of acid secretion (achlorhydria) occurs in gastric carcinoma. However, anacidity occurs only in a small proportion of cases with advanced gastric cancer. Also, not all patients with duodenal ulcer show increased acid output.
5. To measure the amount of acid secreted in a patient with symptoms of peptic ulcer dyspepsia but normal X-ray findings: Excess acid secretion in such cases is indicative of duodenal ulcer. However, hypersecretion of acid does not always occur in duodenal ulcer.
6. To decide the type of surgery to be performed in a patient with peptic ulcer: Raised basal as well as peak acid outputs indicate increased parietal cell mass and need for gastrectomy. Raised basal acid output with normal peak output is an indication for vagotomy.

To assess gastric acid secretion, acid output from the stomach is measured in a fasting state and after injection of a drug which stimulates gastric acid secretion. Basal acid output (BAO) is the amount of hydrochloric acid (HCl) secreted in the absence of any external stimuli (visual, olfactory, or auditory). Maximum acid output (MAO) is the amount of hydrochloric acid secreted by the stomach following stimulation by pentagastrin. MAO is calculated from the first four 15-minute samples after stimulation. Peak acid output (PAO) is calculated from the two highest consecutive 15-minute samples. It indicates greatest possible acid secretory capacity and is preferred over MAO as it is more reproducible. Acidity is estimated by titration.

Microscopic examinations done on fecal sample are shown in Figure 846.1.

Collection of Specimen for Parasites

A random specimen of stool (at least 4 ml or 4 cm³) is collected in a clean, dry, container with a tightly fitting lid (a tin box, plastic box, glass jar, or waxed cardboard box) and transported immediately to the laboratory (this is because trophozoites of Entameba histolytica rapidly degenerate and alter in morphology). About 20-40 grams of formed stool or 5-6 tablespoons of watery stool should be collected. Stool should not be contaminated with urine, water, soil, or menstrual blood. Urine and water destroy trophozoites; soil will introduce extraneous organisms and also hinder proper examination. Parasites are best detected in warm, freshly passed stools and therefore stools should be examined as early as possible after receipt in the laboratory (preferably within 1 hour of collection). If delay in examination is anticipated, sample may be refrigerated. A fixative containing 10% formalin (for preservation of eggs, larvae, and cysts) or polyvinyl alcohol (for preservation of trophozoites and cysts, and for permanent staining) may be used if specimen is to be transported to a distant laboratory.

One negative report for ova and parasites does not exclude the possibility of infection. Three separate samples, collected at 3-day intervals, have been recommended to detect all parasite infections.

Patient should not be receiving oily laxatives, antidiarrheal medications, bismuth, antibiotics like tetracycline, or antacids for 7 days before stool examination. Patient should not have undergone a barium swallow examination.

In the laboratory, macroscopic examination is done for consistency (watery, loose, soft or formed) (Figure 846.2), color, odor, and presence of blood, mucus, adult worms or segments of tapeworms.

Trophozoites are most likely to be found in loose or watery stools or in stools containing blood and mucus, while cysts are likely to be found in formed stools. Trophozoites die soon after being passed and therefore such stools should be examined within 1 hour of passing. Examination of formed stools can be delayed but should be completed on the same day.

Color/Appearance of Fecal Specimens

• Brown: Normal
• Black: Bleeding in upper gastrointestinal tract (proximal to cecum), Drugs (iron salts, bismuth salts, charcoal)
• Red: Bleeeding in large intestine, undigested tomatoes or beets
• Clay-colored (gray-white): Biliary obstruction
• Silvery: Carcinoma of ampulla of Vater
• Watery: Certain strains of Escherichia coli, Rotavirus enteritis, cryptosporidiosis
• Rice water: Cholera
• Unformed with blood and mucus: Amebiasis, inflammatory bowel disease
• Unformed with blood, mucus, and pus: Bacillary dysentery
• Unformed, frothy, foul smelling, which float on water: Steatorrhea.

Preparation of Slides

After receipt in the laboratory, saline and iodine wet mounts of the sample are prepared (Figure 846.3).

A drop of normal saline is placed near one end of a glass slide and a drop of Lugol iodine solution is placed near the other end. A small amount of feces (about the size of a match-head) is mixed with a drop each of saline and iodine using a wire loop, and a cover slip is placed over each preparation separately. If the specimen contains blood or mucus, that portion should be included for examination (trophozoites are more readily found in mucus). If the stools are liquid, select the portion from the surface for examination.

Saline wet mount is used for demonstration of eggs and larvae of helminths, and trophozoites and cysts of protozoa. It can also detect red cells and white cells. Iodine stains glycogen and nuclei of the cysts. The iodine wet mount is useful for identification of protozoal cysts. Trophozoites become non-motile in iodine mounts. A liquid, diarrheal stool can be examined directly without adding saline.

Concentration Procedure

Concentration of fecal specimen is useful if very small numbers of parasites are present. However, in concentrated specimens, amebic trophozoites can no longer be detected since they are destroyed. If wet mount examination is negative and there is clinical suspicion of parasitic infection, fecal concentration is indicated. It is used for detection of ova, cysts, and larvae of parasites.

Various concentration methods are available; the choice depends on the nature of parasites to be identified and the equipment/reagent available in a particular laboratory. Concentration techniques are of two main types:

• Sedimentation techniques: Ova and cysts settle at the bottom. However, excessive fecal debris may make the detection of parasites difficult. Example: Formolethyl acetate sedimentation procedure.
• Floatation techniques: Ova and cysts float on surface. However, some ova and cysts do not float at the top in this procedure. Examples: Saturated salt floatation technique and zinc sulphate concentration technique.

The most commonly used sedimentation method is formol-ethyl acetate concentration method since: (i) it can detect eggs and larvae of almost all helminths, and cysts of protozoa, (ii) it preserves their morphology well, (iii) it is rapid, and (iv) risk of infection to the laboratory worker is minimal because pathogens are killed by formalin.

In this method, fecal suspension is prepared in 10% formalin (10 ml formalin + 1 gram feces). This suspension is then passed through a gauze filter till 7 ml of filtered material is obtained. To this, ethyl acetate (3 ml) is added and the mixture is centrifuged for 1 minute. Eggs, larvae, and cysts sediment at the bottom of the centrifuge tube (Figure 846.4). Above this deposit, there are layers of formalin, fecal debris, and ether. Fecal debris is loosened with an applicator stick and the supernatant is poured off. One drop of sediment is placed on one end of a glass slide and one drop is placed at the other end. One of the drops is stained with iodine, cover slips are placed, and the preparation is examined under the microscope.

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Stool Examination for Ova and Parasites

Parasites are best detected in warm, freshly passed stools and therefore stools should be examined as early as possible after receipt in the laboratory (preferably within 1 hour of collection). Read more...

Classification of Intestinal Parasites of Humans

Intestinal parasites of humans are classified into two main kingdoms: protozoa and metazoa (helminths) (Figure 846.5).

Chemical examination of feces is usually carried out for the following tests (Figure 845.1):

• Occult blood
• Excess fat excretion (malabsorption)
• Urobilinogen
• Reducing sugars
• Fecal osmotic gap
• Fecal pH

Test for Occult Blood in Stools

Presence of blood in feces which is not apparent on gross inspection and which can be detected only by chemical tests is called as occult blood. Causes of occult blood in stools are:

1. Intestinal diseases: hookworms, amebiasis, typhoid fever, ulcerative colitis, intussusception, adenoma, cancer of colon or rectum.
2. Gastric and esophageal diseases: peptic ulcer, gastritis, esophageal varices, hiatus hernia.
3. Systemic disorders: bleeding diathesis, uremia.
4. Long distance runners.

Occult blood test is recommended as a screening procedure for detection of asymptomatic colorectal cancer. Yearly examinations should be carried out after the age of 50 years. If the test is positive, endoscopy and barium enema are indicated.

Tests for detection of occult blood in feces: Many tests are available which differ in their specificity and sensitivity. These tests include tests based on peroxidase-like activity of hemoglobin (benzidine, orthotolidine, aminophenazone, guaiac), immunochemical tests, and radioisotope tests.

Tests Based on Peroxidase-like Activity of Hemoglobin

Principle: Hemoglobin has peroxidase-like activity and releases oxygen from hydrogen peroxide. Oxygen molecule then oxidizes the chemical reagent (benzidine, orthotolidine, aminophenazone, or guaiac) to produce a colored reaction product.

Benzidine and orthotolidine are carcinogenic and are no longer used. Benzidine test is also highly sensitive and false-positive reactions are common. Since bleeding from the lesion may be intermittent, repeated testing may be required.

Causes of False-positive Tests

1. Ingestion of peroxidase-containing foods like red meat, fish, poultry, turnips, horseradish, cauliflower, spinach, or cucumber. Diet should be free from peroxidase-containing foods for at least 3 days prior to testing.
2. Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons.

Causes of False-negative Tests

1. Foods containing large amounts of vitamin C.
2. Conversion of all hemoglobin to acid hematin (which has no peroxidase-like activity) during passage through the gastrointestinal tract.

Immunochemical Tests

These tests specifically detect human hemoglobin. Therefore there is no interference from animal hemoglobin or myoglobin (e.g. meat) or peroxidase-containing vegetables in the diet.

The test consists of mixing the sample with latex particles coated with anti-human haemoglobin antibody, and if agglutination occurs, test is positive. This test can detect 0.6 ml of blood per 100 grams of feces.

Radioisotope Test Using ⁵¹Cr

In this test, 10 ml of patient’s blood is withdrawn, labeled with ⁵¹Cr, and re-infused intravenously. Radioactivity is measured in fecal sample and in simultaneously collected blood specimen. Radioactivity in feces indicates gastrointestinal bleeding. Amount of blood loss can be calculated. Although the test is sensitive, it is not suitable for routine screening.

Apt test: This test is done to decide whether blood in the vomitus or in the feces of a neonate represents swallowed maternal blood or is the result of bleeding in the gastrointestinal tract. The test was devised by Dr. Apt and hence the name. The baby swallows blood during delivery or during breastfeeding if nipples are cracked. Apt test is based on the principle that if blood is of neonatal origin it will contain high proportion of hemoglobin F (Hb F) that is resistant to alkali denaturation. On the other hand, maternal blood mostly contains adult hemoglobin or Hb A that is less resistant.

Test for Malabsorption of Fat

Dietary fat is absorbed in the small intestine with the help of bile salts and pancreatic lipase. Fecal fat mainly consists of neutral fats (unsplit fats), fatty acids, and soaps (fatty acid salts). Normally very little fat is excreted in feces (<7 grams/day in adults). Excess excretion of fecal fat indicates malabsorption and is known as steatorrhea. It manifests as bulky, frothy, and foul-smelling stools, which float on the surface of water.

Causes of Malabsorption of Fat

1. Deficiency of pancreatic lipase (insufficient lipolysis): chronic pancreatitis, cystic fibrosis.
2. Deficiency of bile salts (insufficient emulsification of fat): biliary obstruction, severe liver disease, bile salt deconjugation due to bacterial overgrowth in the small intestine.
3. Diseases of small intestine: tropical sprue, celiac disease, Whipple’s disease.

Tests for fecal fat are qualitative (i.e. direct microscopic examination after fat staining), and quantitative (i.e. estimation of fat by gravimetric or titrimetric analysis).

1. Microscopic stool examination after staining for fat: A random specimen of stool is collected after putting the patient on a diet of >80 gm fat per day. Stool sample is stained with a fat stain (oil red O, Sudan III, or Sudan IV) and observed under the microscope for fat globules (Figure 845.2). Presence of ≥60 fat droplets/HPF indicates steatorrhea. Ingestion of mineral or castor oil and use of rectal suppositories can cause problems in interpretation.
2. Quantitative estimation of fecal fat: The definitive test for diagnosis of fat malabsorption is quantitation of fecal fat. Patient should be on a diet of 70-100 gm of fat per day for 6 days before the test. Feces are collected over 72 hours and stored in a refrigerator during the collection period. Specimen should not be contaminated with urine. Fat quantitation can be done by gravimetric or titrimetric method. In gravimetric method, an accurately weighed sample of feces is emulsified, acidified, and fat is extracted in a solvent; after evaporation of solvent, fat is weighed as a pure compound. Titrimetric analysis is the most widely used method. An accurately weighed stool sample is treated with alcoholic potassium hydroxide to convert fat into soaps. Soaps are then converted to fatty acids by the addition of hydrochloric acid. Fatty acids are extracted in a solvent and the solvent is evaporated. The solution of fat made in neutral alcohol is then titrated against sodium hydroxide. Fatty acids comprise about 80% of fecal fat. Values >7 grams/day are usually abnormal. Values >14 grams/day are specific for diseases causing fat malabsorption.

Test for Urobilinogen in Feces

Fecal urobilinogen is determined by Ehrlich’s aldehyde test (see Article “Test for Detection of Urobilinogen in Urine”). Specimen should be fresh and kept protected from light. Normal amount of urobilinogen excreted in feces is 50-300 mg per day. Increased fecal excretion of urobilinogen is seen in hemolytic anemia. Urobilinogen is deceased in biliary tract obstruction, severe liver disease, oral antibiotic therapy (disturbance of intestinal bacterial flora), and aplastic anemia (low hemoglobin turnover). Stools become pale or clay-colored if urobilinogen is reduced or absent.

Test for Reducing Sugars

Deficiency of intestinal enzyme lactase is a common cause of malabsorption. Lactase converts lactose (in milk) to glucose and galactose. If lactase is deficient, lactose is converted to lactic acid with production of gas. In infants this leads to diarrhea, vomiting, and failure to thrive. Benedict’s test or Clinitest™ tablet test for reducing sugars is used to test freshly collected stool sample for lactose. In addition, oral lactose tolerance test is abnormal (after oral lactose, blood glucose fails to rise above 20 mg/dl of basal value) in lactase deficiency. Rise in blood glucose indicates that lactose has been hydrolysed and absorbed by the mucosa. Lactose tolerance test is now replaced by lactose breath hydrogen testing. In lactase deficiency, accumulated lactose in the colon is rapidly fermented to organic acids and gases like hydrogen. Hydrogen is absorbed and then excreted through the lungs into the breath. Amount of hydrogen is then measured in breath; breath hydrogen more than 20 ppm above baseline within 4 hours indicates positive test.

Fecal Osmotic Gap

Fecal osmotic gap is calculated from concentration of electrolytes in stool water by formula 290-2([Na⁺] + [K⁺]). (290 is the assumed plasma osmolality). In osmotic diarrheas, osmotic gap is >150 mOsm/kg, while in secretory diarrhea, it is typically below 50 mOsm/kg. Evaluation of chronic diarrhea is shown in Figure 845.3.

Fecal pH

Stool pH below 5.6 is characteristic of carbohydrate malabsorption.

Tests to Assess Proximal Tubular Function

Renal tubules efficiently reabsorb 99% of the glomerular filtrate to conserve the essential substances like glucose, amino acids, and water.

1. Glycosuria

In renal glycosuria, glucose is excreted in urine, while blood glucose level is normal. This is because of a specific tubular lesion which leads to impairment of glucose reabsorption. Renal glycosuria is a benign condition. Glycosuria can also occur in Fanconi syndrome.

2. Generalized aminoaciduria

In proximal renal tubular dysfunction, many amino acids are excreted in urine due to defective tubular reabsorption.

3. Tubular proteinuria (Low molecular weight proteinuria)

Normally, low molecular weight proteins (β₂ –microglobulin, retinol-binding protein, lysozyme, and α₁ –microglobulin) are freely filtered by glomeruli and are completely reabsorbed by proximal renal tubules. With tubular damage, these low molecular weight proteins are excreted in urine and can be detected by urine protein electrophoresis. Increased amounts of these proteins in urine are indicative of renal tubular damage.

4. Urinary concentration of sodium

If both BUN and serum creatinine are acutely increased, it is necessary to distinguish between prerenal azotemia (renal underperfusion) and acute tubular necrosis. In prerenal azotemia, renal tubules are functioning normally and reabsorb sodium, while in acute tubular necrosis, tubular function is impaired and sodium absorption is decreased. Therefore, in prerenal azotemia, urinay sodium concentration is < 20 mEq/L while in acute tubular necrosis, it is > 20 mEq/L.

5. Fractional excretion of sodium (FE_Na)

Measurement of urinary sodium concentration is affected by urine volume and can produce misleading results. Therefore, to avoid this, fractional excretion of sodium is calculated. This refers to the percentage of filtered sodium that has been absorbed and percentage that has been excreted. Measurement of fractional sodium excretion is a better indicator of tubular absorption of sodium than quantitation of urine sodium alone.

This test is indicated in acute renal failure. In oliguric patients, this is the most reliable means of early distinction between pre-renal failure and renal failure due to acute tubular necrosis. It is calculated from the following formula:

In pre-renal failure this ratio is less than 1%, and in acute tubular necrosis it is more than 1%. In pre-renal failure (due to reduced renal perfusion), aldosterone secretion is stimulated which causes maximal sodium conservation by the tubules and the ratio is less than 1%. In acute tubular necrosis, maximum sodium reabsorption is not possible due to tubular cell injury and consequently the ratio will be more than 1%. Values above 3% are strongly suggestive of acute tubular necrosis.

Tests to Assess Distal Tubular Function

1. Urine specific gravity

Normal specific gravity is 1.003 to 1.030. It depends on state of hydration and fluid intake.

1. Causes of increased specific gravity:
   a. Reduced renal perfusion (with preservation of concentrating ability of tubules),
   b. Proteinuria,
   c. Glycosuria,
   d. Glomerulonephritis.
   e. Urinary tract obstruction.
2. Causes of reduced specific gravity:
   a. Diabetes insipidus
   b. Chronic renal failure
   c. Impaired concentrating ability due to diseases of tubules.

As a test of renal function, it gives information about the ability of renal tubules to concentrate the glomerular filtrate. This concentrating ability is lost in diseases of renal tubules.

Fixed specific gravity of 1.010, which cannot be lowered or increased by increasing or decreasing the fluid intake respectively, is an indication of chronic renal failure.

2. Urine osmolality

The most commonly employed test to evaluate tubular function is measurement of urine/plasma osmolality. This is the most sensitive method for determination of ability of concentration. Osmolality measures number of dissolved particles in a solution. Specific gravity, on the other hand, is the ratio of mass of a solution to the mass of water i.e. it measures total mass of solute. Specific gravity depends on both the number and the nature of dissolved particles while osmolality is exact number of solute particles in a solution. Specific gravity measurement can be affected by the presence of solutes of large molecular weight like proteins and glucose, while osmolality is not. Therefore measurement of osmolality is preferred.

When solutes are dissolved in a solvent, certain changes take place like lowering of freezing point, increase in boiling point, decrease in vapor pressure, or increase of osmotic pressure of the solvent. These properties are made use of in measuring osmolality by an instrument called as osmometer.

Osmolality is expressed as milliOsmol/kg of water.

Urine/plasma osmolality ratio is helpful in distinguishing pre-renal azotemia (in which ratio is higher) from acute renal failure due to acute tubular necrosis (in which ratio is lower). If urine and plasma osmolality are almost similar, then there is defective tubular reabsorption of water.

3. Water deprivation test

If the value of baseline osmolality of urine is inconclusive, then water deprivation test is performed. In this test, water intake is restricted for a specified period of time followed by measurement of specific gravity or osmolality. Normally, urine osmolality should rise in response to water deprivation. If it fails to rise, then desmopressin is administered to differentiate between central diabetes insipidus and nephrogenic diabetes insipidus. Urinary concentration ability is corrected after administration of desmopressin in central diabetes insipidus, but not in nephrogenic diabetes insipidus.

If urine osmolality is > 800 mOsm/kg of water or specific gravity is ≥1.025 following dehydration, concentrating ability of renal tubules is normal. However, normal result does not rule out presence of renal disease.

False result will be obtained if the patient is on low-salt, low-protein diet or is suffering from major electrolyte and water disturbance.

4. Water loading antidiuretic hormone suppression test

This test assesses the capacity of the kidney to make urine dilute after water loading.

After overnight fast, patient empties the bladder and drinks 20 ml/kg of water in 15-30 minutes. The urine is collected at hourly intervals for the next 4 hours for measurements of urine volume, specific gravity, and osmolality. Plasma levels of antidiuretic hormone and serum osmolality should be measured at hourly intervals.

Normally, more than 90% of water should be excreted in 4 hours. The specific gravity should fall to 1.003 and osmolality should fall to < 100 mOsm/kg. Plasma level of antidiuretic hormone should be appropriate for serum osmolality. In renal function impairment, urine volume is reduced (<80% of fluid intake is excreted) and specific gravity and osmolality fail to decrease. The test is also impaired in adrenocortical insufficiency, malabsorption, obesity, ascites, congestive heart failure, cirrhosis, and dehydration.

This test is not advisable in patients with cardiac failure or kidney disease. If there is failure to excrete water load, fatal hyponatremia can occur.

5. Ammonium chloride loading test (Acid load test)

Diagnosis of renal tubular acidosis is usually considered after excluding other causes of metabolic acidosis. This test is considered as a ‘gold standard’ for the diagnosis of distal or type 1 renal tubular acidosis. Urine pH and plasma bicarbonate are measured after overnight fasting. If pH is less than 5.4, acidifying ability of renal tubules is normal. If pH is greater than 5.4 and plasma bicarbonate is low, diagnosis of renal tubular acidosis is confirmed. In both the above cases, further testing need not be performed. In all other cases in which neither of above results is obtained, further testing is carried out. Patient is given ammonium chloride orally (0.1 gm/kg) over 1 hour after overnight fast and urine samples are collected hourly for next 6-8 hours. Ammonium ion dissociates into H⁺ and NH₃. Ammonium chloride makes blood acidic. If pH is less than 5.4 in any one of the samples, acidifying ability of the distal tubules is normal.

1. Pre-renal azotemia: shock, congestive heart failure, salt and water depletion
2. Renal azotemia: impairment of renal function
3. Post-renal azotemia: obstruction of urinary tract
4. Increased rate of production of urea:
   • High protein diet
   • Increased protein catabolism (trauma, burns, fever)
   • Absorption of amino acids and peptides from a large gastrointestinal hemorrhage or tissue hematoma

1. Diacetyl monoxime urea method: This is a direct method. Urea reacts with diacetyl monoxime at high temperature in the presence of a strong acid and an oxidizing agent. Reaction of urea and diacetyl monoxime produces a yellow diazine derivative. The intensity of color is measured in a colorimeter or spectrophotometer.
2. Urease- Berthelot reaction: This is an indirect method. Enzyme urease splits off ammonia from the urea molecule at 37°C. Ammonia generated is then reacted with alkaline hypochlorite and phenol with a catalyst to produce a stable color (indophenol). Intensity of color produced is then measured in a spectrophotometer at 570 nm.

• It is produced from muscles at a constant rate and its level in blood is not affected by diet, protein catabolism, or other exogenous factors;
• It is not reabsorbed, and very little is secreted by tubules.

1. Pre-renal, renal, and post-renal azotemia
2. Large amount of dietary meat
3. Active acromegaly and gigantism

1. Pregnancy
2. Increasing age (reduction in muscle mass)

1. Jaffe’s reaction (Alkaline picrate reaction): This is the most widely used method. Creatinine reacts with picrate in an alkaline solution to produce spectrophotometer at 485 nm. Certain substances in plasma (such as glucose, protein, fructose, ascorbic acid, acetoacetate, acetone, and cephalosporins) react with picrate in a similar manner; these are called as non-creatinine chromogens (and can cause false elevation of serum creatinine level). Thus ‘true’ creatinine is less by 0.2 to 0.4 mg/dl when estimated by Jaffe’s reaction.
2. Enzymatic methods: These methods use enzymes that cleave creatinine; hydrogen peroxide produced then reacts with phenol and a dye to produce a colored product, which is measured in a spectrophotometer.

1. Increased BUN with normal serum creatinine:
   • Pre-renal azotemia (reduced renal perfusion)
   • High protein diet
   • Increased protein catabolism
   • Gastrointestinal hemorrhage
2. Increase of both BUN and serum creatinine with disproportionately greater increase of BUN:
   • Post-renal azotemia (Obstruction to the outflow of urine)
   Obstruction to the urine outflow causes diffusion of urinary urea back into the blood from tubules because of backpressure.

• Acute tubular necrosis
• Low protein diet, starvation
• Severe liver disease

Glomerular filtration rate refers to the rate in ml/min at which a substance is cleared from the circulation by the glomeruli. The ability of the glomeruli to filter a substance from the blood is assessed by clearance studies. If a substance is not bound to protein in plasma, is completely filtered by the glomeruli, and is neither secreted nor reabsorbed by the tubules, then its clearance rate is equal to the glomerular filtration rate (GFR).

Renal biopsy refers to obtaining a small piece of kidney tissue for microscopic examination. Percutaneous renal biopsy was first performed by Alwall in 1944.