Articles on Medical Diseases and Conditions

Tag: Serum

Diseases of Mineral Metabolism

Wilson’s disease (hepatolenticular degeneration). Wilson’s disease is a familial disorder of copper metabolism transmitted as an autosomal recessive trait. It most often becomes manifest between ages 8 and 30 years; symptoms usually do not develop before age 6 years. About 30%-50% of patients initially develop hepatic symptoms, about 30%-40% begin with neurologic symptoms, and about 20%-30% initially are said to have psychiatric abnormalities such as schizophrenia. A few patients develop a Coombs’-negative hemolytic anemia. Children are more likely to be first seen with hepatic symptoms, although symptoms may occur at any age. In children, these most commonly take the form of chronic hepatitis, although in some patients the test results may resemble acute hepatitis virus hepatitis. A macronodular type of cirrhosis develops later and is usually present in patients with late-stage Wilson’s disease, whether or not there were symptoms of active liver disease. Some patients present with minimally active or with nonactive cirrhosis. Neurologic symptoms typically originate in the basal ganglia area (lentiform nucleus) of the brain and consist of varying degrees of incoordination, tremor, spasticity, rigidity, and dysarthria. There may also be a peculiar flapping tremor. Some young or middle-aged adults develop premature osteoarthritis, especially in the knees.

Wilson’s disease is characterized by inability of the liver to manufacture normal quantities of ceruloplasmin, an alpha-2 globulin that transports copper. For reasons not entirely understood, excessive copper is deposited in various tissues, eventually producing damage to the basal ganglia of the brain and to the liver. The kidney is also affected, leading to aminoaciduria, and copper is deposited in the cornea, producing a zone of discoloration called the Kayser-Fleischer ring.

Clinical diagnosis. The triad of typical basal ganglia symptoms, Kayser-Fleischer ring, and hepatic cirrhosis is virtually diagnostic. However, many patients do not have the textbook picture, especially in the early stages. The Kayser-Fleischer ring is often grossly visible but in many cases can be seen only by slit lamp examination. All patients with neurologic symptoms are said to have the Kayser-Fleischer ring as well as about 50% (range, 27%-93%) of those with hepatic symptoms. The Kayser-Fleischer ring is present in only about 20% (range, 0%-37%) of asymptomatic patients detected during family study investigation or at the beginning of symptoms from hepatic disease without neurologic findings. Overall, about 25% of patients (range, 22%-33%) do not have a demonstrable Kayser-Fleischer ring at the time of diagnosis. Patients with primary biliary cirrhosis or, occasionally, other types of chronic cholestatic liver disease may develop a corneal abnormality identical to the Kayser-Fleischer ring.

Plasma ceruloplasmin assay. Laboratory studies may be of value in diagnosis, especially in the preclinical or early stages. Normally, about 90%-95% of serum copper is bound to ceruloplasmin, one of the alpha-2 globulins. The primary excretion pathway for serum copper is through bile. The serum ceruloplasmin level is low from birth in 95% (range, 90%-96%) of homozygous patients, and is considered the best screening test for Wilson’s disease. About 10% (range, 6%-20%) of Wilson’s disease heterozygotes have decreased serum ceruloplasmin. However, normal newborn infants usually have decreased ceruloplasmin levels, and the test is not considered reliable until 3-6 months of age. Although a normal ceruloplasmin level (over 20 mg/100 ml; 200 mg/L) is usually interpreted as excluding Wilson’s disease, about 5% (range, 4%-10%) of homozygous Wilson’s disease patients have values greater than 20 mg/100 ml. This is more likely to be found in younger children and in those with hepatic disease. Estrogen therapy, pregnancy, active liver disease of various etiologies, malignant lymphoma, and occasionally various acute inflammatory conditions (since ceruloplasmin is one of the “acute reaction” proteins) can raise ceruloplasmin levels in variable numbers of cases. Smoking is reported to raise ceruloplasmin levels about 15%-30%. Although a decreased ceruloplasmin level is usually considered suggestive of Wilson’s disease, about 5% of normal persons may have values less than 20 mg/100 ml (200 mg/L), and values may be decreased in hereditary tyrosinemia, Menke’s kinky hair syndrome, the nephrotic syndrome, malabsorption syndromes such as sprue, and in various liver diseases (about 20% of cases in one study. However, it is possible that some patients with liver disease and decreased ceruloplasmin levels actually have Wilson’s disease).

Liver biopsy has also been used for diagnosis. The microscopic findings are not specific, and most often consist of either macronodular cirrhosis (often with some fatty change and occasionally with Mallory bodies) or chronic active hepatitis (10%-15% of patients with Wilson’s disease). The most typical finding is increased hepatic copper content by special stains (or tissue analysis, if available). For histologic staining of copper, fixation of the biopsy specimen in alcohol rather than the routine fixatives is recommended. Here again, it is advisable to wait 6-12 weeks after birth. Increased hepatic copper content is not specific for Wilson’s disease, since some degree of copper increase has been reported to occur in some patients with postnecrotic cirrhosis due to hepatitis virus hepatitis, in patients with primary biliary cirrhosis, and occasionally in patients with other chronic cholestatic syndromes. Also, increased hepatic copper content is not present in all patients with Wilson’s disease, especially in small-needle biopsy specimens.

Serum and urine copper. Total serum copper levels are decreased in 85%-90% of Wilson’s disease patients. However, serum copper not bound to serum ceruloplasmin is usually normal or increased. Twenty-four-hour urine copper excretion in symptomatic Wilson’s disease is increased in 90% of patients. However, 24-hour copper excretion is often normal in presymptomatic patients. Increased urine copper excretion is not specific for Wilson’s disease and may be found in various types of cirrhosis, especially those with some degree of cholestasis and in 10%-30% of chronic active hepatitis patients. However, these conditions usually have normal or elevated serum ceruloplasmin levels.

DNA probes. The gene affected in Wilson’s disease has been found on the long arm of chromosome 13, close to the gene responsible for retinoblastoma. DNA linkage probes for Wilson’s disease have been reported. In some cases, the retinoblastoma probe has been used.

Other laboratory abnormalities. Besides abnormalities in copper metabolism, over 50% of patients (78% in one study) have a low serum uric acid level, a finding that could arouse suspicion of Wilson’s disease if supporting evidence is present. Other laboratory findings that may be encountered in some patients are low serum phosphorus levels, thrombocytopenia (about 50%; range, 22%-82%, due to cirrhosis with secondary hypersplenism), aminoaciduria, glucosuria, and uricosuria. A Coombs’-negative hemolytic anemia occurs in a few patients.

Hemochromatosis. Hemochromatosis is an uncommon disease produced by idiopathic excess iron absorption from the GI tract, which leads to excess deposition of iron in various tissues, especially the liver. There still is dispute as to which iron storage diseases should be included within the term hemochromatosis. In this discussion, hemochromatosis refers to the hereditary iron storage disorder and hemosiderosis to nonhereditary (secondary) forms. Hemochromatosis is transmitted as an autosomal recessive trait with the gene being located on the short arm of chromosome 6 close to the class I histocompatibility antigen (HLA) locus. Males are affected more often than females (3:2 in one series), and males seem overall to have more severe disease than females. HLA-A3 antigen is present in 70%-80% of patients (vs. 20%-30% in the normal population).

Clinical onset of the disease is usually between ages 40 and 60 years. Signs, symptoms, and laboratory abnormalities depend on the stage of disease and (probably) whether there is also a significant degree of alcohol intake. Cirrhosis, diabetes mellitus, and bronze skin pigmentation form a classic triad diagnostic of hemochromatosis. However, this triad is a late manifestation, and in one study including more early cases it was present in less than 10% of the patients. The most frequent symptom is joint pain (47%-57% of patients; 50%-75% in patients with severe disease), which can be confused with rheumatoid arthritis. Hepatomegaly is present in 54%-93% of patients, cirrhosis on liver biopsy in 57%-94%, heart failure in 0%-35%, hypogonadism (in males) in 18%-61%, skin pigmentation in 51%-85% (not really noticeable in many patients), and clinically evident diabetes in 6%-72%. Alcoholism (15%-50%) or poor nutrition was frequent in some series. Hepatoma has been reported to develop in 15%-30% of patients.

Laboratory findings include the expected blood glucose abnormalities of diabetes (chapter 28) in those patients with overt diabetes, and decreased glucose tolerance in some of those without clinical diabetes. AST levels are elevated in 46%-54% of cases, reflecting active liver cell involvement. In one series, AST, alkaline phosphatase (ALP), and gamma-glutamyltransferase were normal or only mildly elevated unless the patient was alcoholic.

Laboratory iron studies. The body iron abnormality is manifested by actual or relative increase in serum iron levels and decrease in total iron-binding capacity (TIBC), producing increased saturation (% saturation) of the TIBC. In addition, hemosiderin very often can be demonstrated in the urine sediment by iron stains. The most sensitive laboratory test for hemochromatosis is percent saturation of TIBC (or of transferrin), which is greater than 60% (reference range, 16%-50%) in over 90% of male homozygotes and the 60% of females who have iron loading but which misses the 40% of females who do not have iron loading. Transferrin saturation of 50% detects most males or females with or without iron loading. Therefore, it has been proposed that the screening cutoff point should be 60% for males and 50% for females. Serum iron level is increased in more than 80% of patients and serum ferritin level is increased in more than 72% of patients; both of these tests are usually abnormal in affected males but much more variable in females. However, in one report about one third of patients with chronic hepatitis B or C also had elevated serum iron, ferritin, and percent saturation, and serum ferritin is often increased by various acute inflammatory conditions. Liver biopsy demonstrates marked deposition of iron in parenchymal cells and frequently reveals cirrhosis.

The most widely used screening test is serum iron. Elevated values raise the question of hemochromatosis. About 2.4% of normal persons are reported to have elevated serum iron values that spontaneously return to the reference range within 1-2 days. The effect of serum diurnal variation and day-to-day variation must be considered. Serum iron levels can also be increased in chronic hepatitis B or C infection (46% of cases in one study) and in hemosiderosis (nonhereditary iron overload) due to blood transfusion, chronic severe hemolytic anemias, sideroblastic anemias, alcoholic cirrhosis, parenteral iron therapy, and considerably increased iron intake. Several other conditions that may be associated with increased serum iron levels are listed in Table 37-2. Various conditions can lower the serum iron level (especially chronic iron deficiency and moderate or severe chronic disease without iron deficiency), and if one of these conditions is superimposed on hemochromatosis, the serum iron level might be decreased sufficiently to reach the reference range area.

As noted earlier, the best screening procedure is percent saturation of transferrin. This is calculated by dividing the serum iron value by the TIBC value. However, like serum iron, increase in percent transferrin saturation is not specific for hemochromatosis, since there are other conditions that decrease percent saturation, especially alcohol-related active cirrhosis. One study found that drawing specimens after an overnight fast considerably decreased false elevation of percent saturation. In addition, there is considerable variation in the literature as to the percent saturation cutoff point that should be used (50%-80%, with the majority using either 50% or 62%). The lower levels increase sensitivity in detecting hemochromatosis; the higher levels eliminate many patients who do not have hemochromatosis.

Definitive diagnosis is made by liver biopsy and measurement of hepatic iron content. Even liver biopsy iron may not differentiate hemochomatosis from hemosiderosis in some cases, and the liver cells of patients with cirrhosis but without demonstrable abnormality of iron metabolism may display some degree of increased iron deposition.

Family member screening. Hemochromatosis rarely becomes clinically evident before age 30, so that screening family members of patients has been advocated to detect unrecognized homozygotes to begin therapy before clinical symptoms develop. One study found that percent transferrin saturation detected about 90% of occult homozygotes, whereas assay of serum iron levels detected about 85% and assay of serum ferritin levels detected about 50%.

December 28, 2009
Thyroid

Thyroid carcinoma seems to have generated a considerable number of misconceptions. About 20% of these tumors are “pure” papillary, about 10% pure follicular, about 50% mixed papillary and follicular, and about 5% (range, 2%–10%) are called medullary. However, the pure papillary carcinoma usually has a few follicular elements if enough histologic sections are made, and the reverse is sometimes true in follicular tumors. In addition, some pathologists classify the tumors according to the predominant element unless the proportions of each element are very similar. If this were done, about 65% would be called papillary and about 20% follicular. There is enough diversity in classification methods to create difficulty in relating pathology reports to statistics in the literature. Papillary and most mixed papillary-follicular carcinomas metastasize primarily to regional lymph nodes. Prognosis is excellent in young adults but less so in older persons. Follicular carcinoma tends to produce hematogenous metastases, most often to lungs and bone. About 15% (range, 4%–30%) of single palpable nodules not selected by thyroid scan or fine-needle aspiration are malignant when excised.

Thyroid radionuclide scan. A major screening test is the thyroid scan. The characteristic appearance of thyroid carcinoma is a single nonfunctioning nodule. A gland that is multinodular on scan has less chance of containing carcinoma than one with a solitary nodule. On occasion, a palpable nodule may represent metastatic carcinoma from another primary site in a lymph node close to the thyroid.

Radionuclide scanning of thyroid nodules can be done with radioactive iodine (RAI) or technetium 99m pertechnetate. Results from comparison studies usually agree, but occasionally carcinomas that appear to have some function on technetium scan but not on iodine scan have been found. About 20% of single thyroid nodules without demonstrable function on scan are malignant (literature range, 3%–58%). About 6% of nodules with some function (reduced, but present) and about 6%–8% of nodules with apparent normal function (literature range, 0%–38%) are reported to be malignant. In some of these cases, normal thyroid tissue above or below the nodule creates a false impression of nodule function. Hyperactive nodules are very rarely malignant, although occasionally a malignancy is found unexpectedly in the same gland.

A minority of investigators believe that radioiodine or technetium scanning is not helpful in evaluation of thyroid nodules for possible malignancy. As noted previously, a single nodule without demonstrable function on scan has roughly a 20% chance of malignancy, which means that 80% of such nodules will be falsely positive for malignancy. On the other hand, some reports indicate that 6%–8% of nodules with apparently normal function may actually be malignant and thus represent false negative results. Therefore, some investigators rely on criteria other than thyroid scan to determine which patients with thyroid nodules should receive operative therapy. The criteria that have been used include patient history, characteristics of the nodule on physical examination, fine needle aspiration, or response of the nodule to thyroid hormone suppression. In the suppression test, failure of the nodule to diminish at least 50% in size during 3 months of suppression would increase the chance of malignancy.

A significant number of patients are referred for thyroid scan while thyroid uptake of radionuclide is being suppressed by administration of thyroid hormone or by x-ray contrast media. This frequently produces unsatisfactory or even misleading results.

Thyroid scan to detect thyroid carcinoma metastases. A different problem may arise when thyroid cancer is discovered and patients are referred for scanning to detect metastases, either before or after initial therapy. Unless all of the normal thyroid tissue is removed or is ablated by radioiodine therapy, enough of the scanning dose will be taken up by normal tissue to make such attempts useless in most cases. In addition, replacement thyroid hormone administration must cease for 2-4 weeks before scanning, so that the pituitary will once again produce thyroid-stimulating hormone (TSH), which, in turn, will help stimulate the tumor to take up the radioiodine. The dose of (RAI (1–5 mCi; SI, 0.037–0.185 MBq) for a metastatic tumor scan is more than 10 times the usual thyroid scan dose, and the optimal time to scan is 72 hours after administration of the dose. Some prefer a technetium phosphate bone scan to an iodine 131 (131 I) tumor scan. Most bone metastases detected by iodine are also detected by technetium phosphate, and the remaining thyroid tissue does not have to be ablated. However, a few bone metastases are detected by radioiodine and not by technetium. Lung metastases or recurrent neck tumor would be missed using technetium bone scan agents.

Serum thyroglobulin (TG) assay. Serum thyroglobulin (TG) assay has been advocated to follow patients after treatment of thyroid carcinoma. TG is synthesized by thyroid epithelial cells. It is present in measurable amounts in the serum of normal persons on immunoassay (using antibodies against TG) and is increased following TSH stimulation. Elevated values are found in active thyrotoxicosis (diffuse or nodular), thyroiditis, iodine deficiency, benign thyroid adenomas, and differentiated thyroid carcinomas. Therefore, TG elevation is too nonspecific to use for diagnosis of thyroid carcinoma. In thyroid carcinoma, the TG level is usually elevated in papillary, follicular, and mixed papillary-follicular neoplasms. Some anaplastic thyroid carcinomas produce elevated values and some do not. Medullary carcinomas do not produce measurable serum levels of TG. The TG assay can be used to monitor the progress of differentiated thyroid carcinomas after treatment. The half-life of circulating TG is said to be 8-22 hours, so circulating levels should be absent in 7-14 days after total destruction of all normal thyroid tissue and tumor tissue by surgery. Ablation by radioactive iodine is much more gradual and variable. TG values that are nondetectable or nearly so following therapy signify that no residual thyroid or tumor remains, and future elevations mean tumor recurrence or metastasis. Thyroglobulin values that are within the reference range following therapy could either be tumor or could be remnants of normal thyroid, and a thyroid tumor scan with 131 I is required to differentiate these possibilities.

One advantage of TG monitoring is less need for 131 I scanning. This avoids both additional radiation and the need to temporarily stop thyroxine replacement therapy to perform the scan. Also, occasionally patients with metastases associated with elevated TG levels but not detected on 131 I tumor scan have been reported. Disadvantages include a small number of patients with metastases detected on 131 I tumor scan but TG values within the reference range. This occurs in about 4% of cases (literature range, 0%–63%). TG levels are more likely to be normal with pure papillary tumors or those with only lung metastases. Also, the presence of patient anti-TG autoantibodies may interfere with the TG assay.

Fine-needle aspiration cytology. Fine-needle aspiration of thyroid nodules with cytologic smear examination of the aspirate has been advocated to aid diagnosis and, when possible, to replace surgical biopsy. Results in the literature vary rather widely, partially depending on experience with the technique, patient selection, and method of reporting positive results (for example, “definitely malignant” would detect fewer cases of carcinoma than the combination of “malignant” and “suspicious for malignancy”). Some centers report a false negative rate of less than 5% and a false positive rate of less than 2%. Most hospitals could not expect to achieve such good results. The average false negative rate for malignancy with experienced cytologists is about 5%–10%, and the average reported rate overall is about 10%–15% (literature range, 0%–50%). Follicular carcinoma is more difficult to diagnose than papillary carcinoma. The average false positive rate for experienced cytologists is about 2%–4%, and the average reported rate overall is about 5% (range, 0%–14%). Most pathologists without special interest or extensive experience in fine-needle aspiration cytology are better able to interpret needle tissue biopsy material than thyroid aspiration cytology, because thyroid cytology takes special training and experience. However, well-differentiated follicular carcinoma is difficult to diagnose on needle biopsy as well as on aspiration. Needle biopsy is also useful to diagnose thyroiditis.

Thermography and B-mode ultrasound have been used to help evaluate thyroid nodules for malignancy. Results of thermography to date have been rather disappointing. Ultrasound has been used to differentiate cystic thyroid lesions from solid ones. About 15%–20% of thyroid nodules that fail to concentrate radioactive iodine are cystic. Typical completely cystic lesions are rarely malignant (about 2%; literature range, 0%–14%). Ultrasound accuracy in differentiating pure cystic lesions from solid or mixed cystic-solid lesions is usually quoted as about 95% (80%–100%). The procedure in many clinics is to perform aspiration with cytology on ultrasonically pure cysts.

Medullary carcinoma of the thyroid. Medullary carcinoma constitutes 5% (range, 2%–10%) of thyroid carcinomas. It is derived from certain stromal cells known as “C-cells.” The tumor has an intermediate degree of malignancy. It may occur sporadically or in a hereditary form. The sporadic form comprises 80%–90% of cases and is usually unilateral. The familial variety is transmitted as an autosomal dominant trait, is usually present in both thyroid lobes, and is frequently associated with other neoplasms (phenochromocytoma, mucosal neuromas) as part of MEN II (Sipple Syndrome,Table 33-13) or MEN III. This also includes some degree of association with other endocrine abnormalities, such as parathyroid adenoma and Cushing’s syndrome. The tumor may have a variety of histologic patterns, but the classic form is solid nests of cells that are separated by a stroma containing amyloid. These tumors have aroused great interest, since most secrete abnormal amounts of the hormone calcitonin (thyrocalcitonin). Calcitonin has a calcium-lowering action derived from inhibition of bone resorption; therefore, calcitonin acts as an antagonist to parathyroid hormone. Thyroid C cells produce calcitonin as a normal reaction to the stimulus of hypercalcemia. About 70%–75% of medullary carcinomas produce elevated levels of serum calcitonin; this includes most sporadic (nonfamilial) cases. About 25%–30% of familial medullary carcinoma (MEN type III or IIB) have normal basal calcitonin levels. In patients with normal basal calcitonin levels, elevated calcitonin values can be induced by stimulation with calcium infusion or pentagastrin. Glucagon also stimulates calcitonin secretion but not as effectively. A few medullary carcinomas are reported to secrete serotonin or prostaglandins. About 30% of patients experience diarrhea. Besides medullary thyroid carcinoma, calcitonin secretion has been reported in as many as 60% of patients with bronchogenic carcinoma (small cell and adenocarcinoma tumor types).

Table 33-13 Multiple endocrine neoplasias

December 28, 2009
Male Infertility or Hypogonadism

About 40%-50% of infertility problems are said to be due to dysfunction of the male reproductive system. Male infertility can be due to hormonal etiology (either lack of gonadotropin or suppression of spermatogenesis), nonhormonal factors affecting spermatogenesis, primary testicular disease, obstruction to sperm passages, disorders of sperm motility or viability or presence of antisperm antibodies (see the box on this page). About 30%-40% is reported to be associated with varicocele. Diagnosis and investigation of etiology usually begins with physical examination (with special attention to the normality of the genitalia and the presence of a varicocele), semen analysis (ejaculate quantity, number of sperm, sperm morphology, and sperm motility), and serum hormone assay (testosterone, LH, and FSH).

Some Important Causes of Male Infertility, Classified According to Primary Site of the Defect

Hormonal
Insufficient hypothalamic gonadotropin (hypogonadotropic eunuchoidism)
Insufficient pituitary gonadotropins (isolated LH deficiency [“fertile eunuch”] or pituitary
insufficiency)
Prolactin-secreting pituitary adenoma
Excess estrogens (cirrhosis, estrogen therapy, estrogen-producing tumor)
Excess androgens
Excess glucocorticosteroids
Hypothyroidism
Nonhormonal factors affecting testis sperm production
Varicocele
Poor nutrition
Diabetes mellitus
Excess heat in area of testes
Stress and emotion
Drugs and chemicals
Febrile illnesses
Cryptochism(undescended testis, unilateral or bilateral)
Spinal cord injuries
Primary testicular abnormality
Maturation arrest at germ cell stage
“Sertoli cell only” syndrome
Klinefelter’s syndrome and other congenital sex chromosome disorders
Testicular damage (radiation, mumps orchitis, inflammation, trauma)
Myotonic dystrophy
Posttesticular abnormality
Obstruction of sperm passages
Impaired sperm motility
Antisperm antibodies

Testicular function tests. Male testicular function is based on formation of sperm by testicular seminiferous tubules under the influence of testosterone. FSH is necessary for testicular function because it stimulates seminiferous tubule (Sertoli cell) development. Testosterone secretion by the Leydig cells (interstitial cells) of the testis is necessary for spermatogenesis in seminiferous tubules capable of producing sperm. LH (in males sometimes called “interstitial cell-stimulating hormone”) stimulates the Leydig cells to produce testosterone. Testosterone is controlled by a feedback mechanism whereby testosterone levels regulate hypothalamic secretion of GnRH, which, in turn, regulates pituitary secretion of LH. The adrenals also produce androgens, but normally this is not a significant factor in males. In classic cases, serum levels of testosterone and gonadotropins (LH and FSH) can differentiate between primary testicular abnormality (failure of the testis to respond to pituitary gonadotropin stimulation, either congenital or acquired) and pituitary or hypothalamic dysfunction (either congenital or acquired). In primary gonadal (testis) insufficiency, pituitary gonadotropin levels are usually elevated. Of the various categories of primary gonadal insufficiency, the Klinefelter’s xxy chromosome syndrome in its classic form shows normal FSH and LH gonadotropin levels during childhood, but after puberty both FSH and LH levels are elevated and the serum testosterone level is low. Klinefelter’s syndrome exists in several variants, however, and in some cases LH levels may be within reference range (single LH samples also may be confusing because of the pulsatile nature of LH secretion). Occasionally patients with Klinefelter’s syndrome have low-normal total serum testosterone levels (due to an increase in SHBG levels) but decreased free testosterone levels. Elevated pituitary gonadotropin levels should be further investigated by a buccal smear for sex chromatin to elevate the possibility of Klinefelter’s syndrome. Some investigators may perform a chromosome analysis because of possible inaccuracies in buccal smear interpretation and because some persons with Klinefelter’s syndrome have cell mosaicism rather than the same chromosome pattern in all cells, or a person with findings suggestive of Klinefelter’s syndrome may have a different abnormal chromosome karyotype. In the “Sertoli cell only syndrome,” testicular tubules are abnormal but Leydig cells are normal. Therefore, the serum FSH level is elevated but LH and testosterone levels are usually normal. In acquired gonadal failure (due to destruction of the testicular tubules by infection, radiation, or other agents), the FSH level is often (but not always) elevated, whereas LH and testosterone levels are often normal (unless the degree of testis destruction is sufficient to destroy most of the Leydig cells, a very severe change). In secondary (pituitary or hypothalamic) deficiency, both the pituitary gonadotropin (FSH and LH) and the testosterone levels are low.

Although serum testosterone is normally an indicator of testicular Leydig cell function and, indirectly, of pituitary LH secretion, other factors can influence serum testosterone levels. The adrenal provides much of the precursor steroids for testosterone, and some types of congenital adrenal hyperplasia result in decreased levels of testosterone precursors. Cirrhosis may decrease serum testosterone levels. Increase or decrease of testosterone-binding protein levels may falsely increase or decrease serum testosterone levels, since the total serum testosterone value is being measured, whereas assay of free (unbound) testosterone is not affected.

Stimulation tests. Stimulation tests are available to help determine which organ is malfunctioning in equivocal cases.

1. HCG directly stimulates the testis, increasing testosterone secretion to at least twice baseline values.
2. Clomiphene stimulates the hypothalamus, producing increases in both LH and testosterone levels. The medication is given for 10 days. However, clomiphene does not stimulate LH production significantly in prepubertal children. Serum testosterone and LH values must be close to normal pubertal or adult levels before the test can be performed, and the test can be used only when there is a mild degree of abnormality in the gonadal-pituitary system.
3. The GnRH stimulation test can directly evaluate pituitary capability to produce LH and FSH and can indirectly evaluate testicular hormone-producing function that would otherwise depend on measurement of basal testosterone levels. Normally, pituitary LH stimulates testosterone production from Leydig cells of the testis, and the amount of testosterone produced influences hypothalamic production of GnRH, which controls pituitary LH. When exogenous (test) GnRH is administered, the pituitary is stimulated to produce LH. The release of testosterone from the testis in response to LH stimulation inhibits further LH stimulation to some degree. In most male patients with primary gonadal failure, basal serum LH and FSH become elevated due to lack of inhibitory feedback from the testis. However, some patients may have basal serum LH and FSH levels in the lower part of the population reference range, levels that are indistinguishable from those of some normal persons; although preillness values were higher in these patients, their preillness normal levels are rarely known. In these patients, a GnRH stimulation test may be useful. Some investigators have found that the degree of inhibition of LH production in response to GnRH administration is more sensitive in detecting smaller degrees of testicular hormone production insufficiency than basal LH levels. The decreased testosterone levels decrease feedback inhibition on the hypothalamus and administration of GnRH results in an exaggerated LH or FSH response (markedly elevated LH and FSH levels compared to levels in normal control persons). The test is performed by obtaining a baseline serum specimen for LH and FSH followed by an intravenous (IV) bolus injection of 100 µg of synthetic GnRH. Another serum specimen is obtained 30 minutes later for LH and FSH. Certain factors can influence or interfere with GnRH results. Estrogen administration increases GnRH effect by sensitizing the pituitary, and androgens decrease the effect. Patient sex and age (until adult pulsatile secretion schedule is established) also influence test results. When used as a test for pituitary function, some patients with clinically significant degrees of failure show a normal test result; therefore, only an abnormal result is definitely significant.

Semen analysis. Semen analysis is an essential part of male infertility investigation. Semen findings that are highly correlated with hypofertility or infertility include greatly increased or decreased ejaculate volume, very low sperm counts, greatly decreased sperm motility, and more than 20% of the sperm showing abnormal morphology. However, the World Health Organization (WHO) recently changed its definition of semen morphologic abnormality, and now considers a specimen abnormal if more than 70% of the sperm have abnormal morphology. One particular combination of findings is known as the “stress pattern” and consists of low sperm count, decreased sperm motility, and more than 20% of the sperm being abnormal in appearance (especially with an increased number of tapering head forms instead of the normal oval head). The stress pattern is suggestive of varicocele but can be due to acute febrile illness (with effects lasting up to 60 days), some endocrine abnormalities, and antisperm agents. An abnormal semen analysis should be repeated at least once after 3 weeks and possibly even a third time, due to the large variation within the population and variation of sperm production even within the same person as well as the temporary effects of acute illness. Most investigators recommend that the specimen be obtained 2-3 days after regular intercourse to avoid artifacts that may be produced by prolonged sperm storage. The specimen should be received by the laboratory by 1 hour after it is obtained, during which time it should be kept at room temperature.

Fig. 31-1 Semen analysis: sperm morphology. a, acrosome; b, nucleus (difficult to see); c, post-acrosomal cap; d, neckpiece; e, midpiece (short segment after neckpiece); f, tail. A, normal; B, normal (slightly different head shape); C, tapered head; D, tapered head with acrosome deficiency; E, acrosomal deficiency; F, head vacuole; G, cytoplasmic extrusion mass; H, bent head (bend of 45° or more); I, coiled tail; J, coiled tail; K, double tail; L, pairing phenomenon (sperm agglutination); M, sperm precursors (spermatids); N, bicephalic sperm.

Testicular biopsy. Testicular biopsy may be useful in a minority of selected patients in whom no endocrinologic or other cause is found to explain infertility. Most investigators agree that complete lack of sperm in semen analysis is a major indication for biopsy but disagree on whether biopsy should be done if sperm are present in small numbers. Biopsy helps to differentiate lack of sperm production from sperm passage obstruction and can suggest certain possible etiologies of testicular dysfunction or possible areas to investigate. However, none of the histopathologic findings is specific for any one disease. The report usually indicates if spermatogenesis is seen and, if so, whether it is adequate, whether there are normal numbers of germ cells, and whether active inflammation or extensive scarring is present.

Serum antisperm antibody studies. In patients with no evidence of endocrine, semen, or other abnormality, serum antisperm antibody titers can be measured. These studies are difficult to perform and usually must be done at a center that specializes in reproductive studies or therapy. Serum antisperm antibodies are a common finding after vasectomy, being reported in about 50% of cases (literature range, 30%-70%). The incidence and significance of serum antisperm antibodies in couples with infertility problems are somewhat controversial. There is wide variance of reported incidence (3.3%-79%), probably due to different patient groups tested and different testing methods. In men, one report suggests that serum antibody titer is more significant than the mere detection of antibody. High titers of antisperm antibody in men were considered strong evidence against fertility; low titers were of uncertain significance, and mildly elevated titers indicated a poor but not hopeless prognosis. In women, serum antisperm antibodies are said to be present in 7%-17% of infertility cases but their significance is rather uncertain, since a considerable percentage of these women can become pregnant. Antisperm antibody detected in the cervical mucus of women is thought to be much more important than that detected in serum. A test showing ability of sperm to bind to mannose may be available.

December 24, 2009
Tests of Gonadal Function

The most common conditions in which gonadal function tests are used are hypogonadism in males and menstrual disorders, fertility problems, and hirsutism or virilization in females. The hormones currently available for assistance include lutropin (luteinizing hormone; LH), follitropin (follicle-stimulating hormone; FSH), testosterone, human chorionic gonadotropin (hCG), and gonadotropin-releasing hormone (GnRH).

Gonadal function is regulated by hypothalamic secretion of a peptide hormone known as gonadotropin-releasing hormone (GnRH; also called luteinizing hormone-releasing hormone, LHRH). Secretion normally is in discontinuous bursts or pulses, occurring about every 1.5-2.0 hours (range, 0.7-2.5 hours). GnRH half-life normally is about 2-4 minutes (range, 2-8 minutes). There is little if any secretion until the onset of puberty (beginning at age 8-13 years in females and age 9-14 years in males). Then secretion begins predominately at night but later extends throughout the night into daytime, until by late puberty secretion takes place all 24 hours. GnRH is secreted directly into the circulatory channels between the hypothalamus and pituitary. GnRH causes the basophilic cells of the pituitary to secrete LH and FSH. In males LH acts on Leydig cells of the testis to produce testosterone, whereas FSH stimulates Sertoli cells of the testis seminiferous tubules into spermatogenesis, assisted by testosterone. In females, LH acts on ovarian thecal cells to produce testosterone, whereas FSH stimulates ovarian follicle growth and also causes follicular cells to convert testosterone to estrogen (estradiol). There is a feedback mechanism from target organs to the hypothalamus to control GnRH secretion. In males, testosterone and estradiol inhibit LH secretion at the level of the hypothalamus, and testosterone also inhibits LH production at the level of the pituitary. A hormone called inhibitin, produced by the Sertoli cells of the testis tubules, selectively inhibits FSH at both the hypothalamic and pituitary levels. There appears to be some inhibitory action of testosterone and estradiol on FSH production as well, in some circumstances. In females, there is some inhibitory feedback to the hypothalamus by estradiol.

Luteinizing hormone and follicle-stimulating hormone

Originally FSH and LH were measured together using bioassay methods, and so-called FSH measurements included LH. Immunoassay methods can separate the two hormones. The LH cross-reacts with hCG in some RIA systems. Although this ordinarily does not create a problem, there is difficulty in pregnancy and in patients with certain neoplasms that secrete hCG. Several of the pituitary and placental glycopeptides (TSH, FSH, hCG, LH) are composed of at least two immunologic units, alpha and beta. All of these hormones have the same immunologic response to their alpha fraction but a different response to each beta subunit. Antisera against the beta subunit of hCG eliminate interference by LH, and some investigators have reported success in producing antisera against the beta subunit of LH, which does not detect hCG. There is some cross-reaction between FSH and TSH, but as yet this has not seemed important enough clinically in TSH assay to necessitate substitution of TSH beta subunit antiserum for antisera already available. Assay of FSH in serum is thought to be more reliable than in urine due to characteristics of the antibody preparations available.

Serum luteinizing hormone

LH is secreted following intermittent stimulation by GnRH in pulses occurring at a rate of about 2-4 every 6 hours, ranging from 30% to nearly 300% over lowest values. Therefore, single isolated LH blood levels may be difficult to interpret and could be misleading. It has been suggested that multiple samples be obtained (e.g., four specimens, each specimen collected at a 20-minute interval from another). The serum specimens can be pooled to obtain an average value. FSH and testosterone have a relatively constant blood level in females.

Urine luteinizing hormone

In contrast to serum LH, urine LH is more difficult to obtain, since it requires a 24-hour specimen with the usual problem of complete specimen collection. It also has the disadvantage of artifact due to urine concentration or dilution. The major advantage is averaging of 24-hour secretion, which may prevent misleading results associated with serum assays due to LH pulsatile secretion. Another role for urine LH assay is detection of ovulation during infertility workups. LH is rapidly excreted from plasma into urine, so that a serum LH surge of sufficient magnitude is mirrored in single urine samples. An LH surge precedes ovulation by about 24-36 hours (range, 22-4 hours). Since daily urine specimens are obtained beginning 9-10 days after onset of menstruation (so as not to miss the LH surge preceding ovulation if ovulation should occur earlier than the middle of the patient cycle). It has been reported that the best time to obtain the urine specimen is during midday (11 A.M.-3 P.M.) because the LH surge in serum usually takes place in the morning (5 A.M.-9 A.M.). In one study, the LH surge was detected in 56% of morning urine specimens, 94% of midday specimens, and 88% of evening specimens. In contrast, basal body temperature, another method of predicting time of ovulation, is said to be accurate in only 40%-50% of cases. Several manufacturers have marketed simple immunologic tests for urine LH with a color change endpoint that can be used by many patients to test their own specimens. Possible problems include interference by hCG with some of the LH kits, so that early pregnancy could simulate a LH surge. Since the LH surge usually does not last more than 2 days, positive test results for more than 3 consecutive days suggest some interfering factor. Similar interference may appear in some patients with elevated serum LH levels due to ovarian failure (polycystic ovary [PCO] disease, early menopause, etc.).

Finally, an estimated 10% of patients have more than one urine LH spike, although the LH surge has the greatest magnitude. It may be necessary to obtain serum progesterone or urine pregnanediol glucuronide assays to confirm that elevation of LH values is actually the preovulation LH surge.

Urine pregnanediol

The ovarian corpus luteum formed shortly after ovulation secretes increasing amounts of progesterone. Progesterone or its metabolite pregnanediol glucuronide begins to appear in detectable quantities about 2-3 days after ovulation (3-5 days after the LH surge) and persists until about the middle of the luteal phase that ends in menstruation. Conception is followed by progesterone secretion by the placenta. A negative baseline urine pregnanediol assay before the LH surge followed by a positive pregnanediol test result on at least 1 day of early morning urine specimens obtained 7, 8, and 9 days after the LH surge confirm that the LH surge did occur and was followed by ovulation and corpus luteum formation. Urine specimens are collected in the morning rather than at the LH collection time of midday. At least one manufacturer markets a simple kit for urine pregnanediol assay.

Problems with interpretation of a positive urine pregnanediol glucuronide assay include the possibility that early pregnancy may be present. Also, 5%-10% of patients throughout their menstrual cycle nonovulatory phase have slightly or mildly increased pregnanediol levels compared to the reference range. A urine sample collected before the LH surge can be tested to exclude or demonstrate this phenomenon.

Serum androgens

The most important androgens are dehydroepiandrosterone (DHEA), a metabolite of DHEA called DHEA-sulfate (DHEA-S), androstenedione, and testosterone. DHEA is produced in the adrenal cortex, ovary, and testis (in the adrenal cortex, the precursors of DHEA are also precursors of cortisol and aldosterone, which is not the case in the ovary or testis). DHEA is the precursor of androstenedione, and androstenedione is a precursor both of testosterone and of estrogens (see Fig. 30-2). About 50%-60% of testosterone in normal females is derived from androstenedione conversion in peripheral tissues, about 30% is produced directly by the adrenal, and about 20% is produced by the ovary. DHEA blood levels in females are 3 times androstenedione blood levels and about 10 times testosterone blood levels. In normal males after onset of puberty, testosterone blood levels are twice as high as all androgens combined in females. Androstenedione blood levels in males are about 60% of those in females and about 10%-15% of testosterone blood levels.

Serum testosterone

About 60%-75% of circulating serum testosterone is bound to a beta globulin variously known as sex hormone-binding globulin (SHBG) or as testosterone-binding globulin (TBG, a misleading abbreviation because of its similarity to the more widely used abbreviation representing thyroxine-binding globulin). About 20%-40% of testosterone is bound to serum albumin and 1%-2% is unbound (“free”). The unbound fraction is the only biologically active portion. Serum assays for testosterone measure total testosterone (bound plus unbound) values. Special techniques to assay free testosterone are available in some reference laboratories and in larger medical centers. Circulating androstenedione and DHEA are bound to albumin only.

Several conditions can affect total serum testosterone levels by altering the quantity of SHBG without affecting free testosterone. Factors that elevate SHBG levels include estrogens (estrogen-producing tumor, oral contraceptives, or medication), cirrhosis, hyperthyroidism, and (in males) decreased testis function or testicular feminization syndrome. Conditions that decrease SHBG levels include androgens and hypothyroidism.

There is a relatively small diurnal variation of serum testosterone in men, with early morning levels about 20% higher than evening levels. There is little diurnal change in women. Serum levels of androstenedione in men or women are about 50% higher in the early morning than in the evening.

In males, testosterone production is regulated by a feedback mechanism with the hypothalamus and pituitary. The hypothalamus produces gonadotropin-releasing hormone (GnRH; also called LHRH), which induces the pituitary to secrete LH (and FSH). LH, in turn, stimulates the Leydig cells of the testis to secrete testosterone.

Serum estrogens

There are three major estrogens: estrone ((E1), estradiol (estradiol-17b; E2), and estriol (E3). All of the estrogens are ultimately derived from androgenic precursors (DHEA and androstenedione), which are synthesized by the adrenal cortex, ovaries, or testis. The adrenal is unable to convert androgens to estrogens, so estrogens are directly produced in the ovary or testis (from precursors synthesized directly in those organs) or are produced in nonendocrine peripheral tissues such as the liver, adipose tissue, or skin by conversion of androgenic precursors brought by the bloodstream from one of the organs of androgen synthesis. Peripheral tissue estrogens are derived mainly from adrenal androgens. Estradiol is the predominant ovarian estrogen. The ovary also secretes a much smaller amount of estrone. The primary pathway of estradiol synthesis is from androstenedione to estrone and then from estrone to estradiol by a reversible reaction. This takes place in the ovary and to a much lesser extent in peripheral conversion of testosterone to estradiol. Estrone is produced directly from androstenedione (mostly in peripheral tissues) and to a lesser extent in the ovary from already formed estradiol by the reversible reaction. Estriol in nonpregnant women is formed as a metabolite of estradiol or estrone. In pregnancy, estriol is synthesized by the placenta from DHEA (actually from DHEA-S) derived from the fetal adrenals. This is a different pathway from the one in nonpregnant women. Estriol is the major estrogen of the placenta. The placenta also produces some estradiol, derived from both fetal and maternal precursors. Nonendocrine peripheral tissues (liver, skin, fat) synthesize a small amount of estrone and estradiol in pregnancy, mainly from adrenal precursors.

In females, there is a feedback mechanism for estradiol production involving the ovaries, hypothalamus, and pituitary. As already mentioned, the hypothalamus produces GnRH. The GnRH is secreted in a pulsatile manner about every 70-90 minutes and is excreted by glomerular filtration. The GnRH stimulates FSH and LH secretion by the pituitary. Some investigators believe there may be a separate (undiscovered) factor that regulates FSH secretion. The FSH acts on the ovarian follicles to stimulate follicle growth and development of receptors to the action of LH. The LH stimulates the follicles to produce estradiol.

Estradiol values can be measured in serum, and estriol values can be measured in serum or urine by immunoassay. Total estrogen values are measured in urine by a relatively old but still useful procedure based on the Kober biochemical reaction. All of the estrogens can be assayed by gas chromatography. The DHEA and androstenedione values can also be measured by special techniques, but these are available only in large reference laboratories.

December 24, 2009
Thyroid Tests in Hypothyroidism

Serum thyroxine. Thyroxine is frequently used as the major screening test for hypothyroidism, since the T4 level is low in most cases. There is some overlap between hypothyroid patients and normal persons in the lower part of the T4 reference range, since persons with mild, early, or subclinical disease may be inadvertently included in groups of clinically normal persons used to establish the reference range. There is some evidence that nearly all hypothyroid patients within euthyroid population reference limits have T4 values in the lower 50% of the reference range, so that T4 values in the upper half of the reference range are generally reliable in excluding hypothyroidism. Laboratory error, of course, must be considered if the laboratory result does not conform to the clinical picture. If the patient specimens are kept at room temperature for more than 48-72 hours, as might happen when they are sent by mail, increase in fatty acids during transit may falsely increase T4values when competitive binding (displacement) T4 methods rather than radioimmunoassay methods are used. Conditions that alter T4 results, such as TBG changes, non thyroidal illness, and certain medications must be remembered. Some endocrinologists are using TSH assay as a screening test instead of T4.

Triiodothyronine-radioimmunoassay. T3-RIA has not proved very useful in the diagnosis of hypothyroidism. The majority of reports indicate that one fourth to one third of hypothyroid patients have T3-RIA values within normal range. In some cases, typically in Hashimoto’s thyroiditis or after treatment of hyperthyroidism with radioactive iodine, it is thought that normal-range T3-RIA values are due to preferential secretion of T3 in what has been called the “failing gland syndrome.” Test alterations due to non thyroidal illness, age-related decrease, and TBG alterations further complicate interpretation.

Thyroid hormone-binding ratio. The THBR (T3U) test is another test that has not been very helpful in screening for myxedema because of substantial overlap with the euthyroid reference range. The major benefit from its use in possible hypothyroidism is for detection of TBG abnormality.

Serum thyrotropin (TSH) assay. Serum TSH levels are increased in the great majority of patients with primary hypothyroidism, and serum TSH assay is currently the most useful first-line confirmatory test. Since secondary hypothyroidism (pituitary failure) is uncommon and dysfunction due to hypothalamic etiology is rare, TSH assay has also been advocated as a screening test. Until recently TSH assay had not found wider use in screening for thyroid disease in general because of considerable overlap in the low range between hyperthyroid and euthyroid persons. This occurred because in most TSH assay kits the lower limit of the euthyroid reference range is relatively close to zero. In addition, these kits had relatively poor sensitivity in the low range, so that it was difficult to separate hyperthyroid values, which typically are subnormal, from zero on one hand and lower limit of normal on the other. Some euthyroid lowerormal specimens demonstrated the same problem. Therefore, TSH assay was restricted mostly to diagnosis of hypothyroidism. As mentioned earlier, several ultra sensitive TSH kits have recently become available that have adequate sensitivity in the low range to reliably separate decreased TSH values from low normal values. The ultra sensitive TSH is now being advocated by some investigators as the best single screening test for thyroid disease in general. But as I mentioned earlier, in my experience, at present all ultra sensitive TSH kits are not equally reliable. The TSH levels may be increased, usually (but not always) to mild degree, in some clinically euthyroid patients with a variety of conditions (see the box). When the TSH level is elevated in conditions other than primary hypothyroidism, TSH values are usually less than twice the upper reference range limit. However, sometimes they may be as high as 3 times the upper limit and occasionally even higher.

Primary hypothyroidism constitutes 95% or more of hypothyroid cases. The TSH assay in conjunction with the serum T4 assay is sufficient for diagnosis in the great majority of these patients (decreased T4 level with TSH level elevated more than twice and preferably more than 3 times the upper reference limit). In equivocal cases, a TRH test may be useful either to confirm primary hypothyroidism or to differentiate primary from secondary or tertiary etiology. As noted in the section on the TRH test, usefulness of the TRH test may be limited in severe non thyroidal illness. In those circumstances, a TSH stimulation test might be useful.

It has been reported that there are several subgroups of patients with primary hypothyroidism, ranging from those with classic symptoms and markedly elevated TSH values to those with milder symptoms and only mildly elevated TSH values to those with equivocal or single symptoms and T4 and TSH values remaining within population reference range and only the TRH test result abnormal.

About 5%-10% of patients referred to psychiatrists with symptoms of mood depression (“melancholia”) have been reported to have laboratory evidence of hypothyroidism. This evidence ranges from decreased T4and elevated TSH levels to an exaggerated TRH test response as the only abnormality.

In secondary hypothyroidism, the thyroid is normal but malfunction occurs in either the hypothalamus or the pituitary. Typically, both T4 (or FT4) and TSH values are decreased. In a few cases, the TSH value is within normal range; the TSH however is structurally defective and cannot stimulate the thyroid normally.

Thyrotropin-releasing hormone (TRH) test. A more complete discussion of the TRH test is located in the early part of this chapter. The TRH test has been mentioned as a confirmatory test for hypothyroidism. The TRH test has also been used to differentiate secondary from tertiary hypothyroidism. A significant increase in TSH after administration of TRH should theoretically suggest a hypothalamic rather than pituitary etiology for nonprimary hypothyroidism. Unfortunately, 40% of TSH hyposecretors of pituitary origin demonstrate adequate response to TRH stimulation. Therefore, only proof of pituitary hyposecretion by a poor response is considered sufficiently reliable for definite diagnosis. Even a poor response may not be reliable in the presence of severe non thyroidal illness.

December 20, 2009
Serum Fructosamine Assay

Besides Hb A, albumin and various globulins may undergo nonenzymatic glycosylation. In contrast to hemoglobin, which has a serum half-life of about 60 days, albumin has a half-life of about 17-20 days, and total protein (roughly one half albumin and one half globulins) has a half-life of about 30 days. Either glycosylated albumin or glycosylated total protein can be assayed, but most laboratories assay total protein using the fructosamine procedure. This does not involve the sugar fructose and is based on biochemical reaction with glucose bound to protein with a ketoamine linkage, most often using nitro blue tetrazolium as the reagent. Serum fructosamine assay results indicate average glycosylation within the preceding 2-week time period (range, 1-3 weeks). This time period is considerably shorter than that of glycoHb but substantially longer than that for labile hemoglobin glycosylation. Drawbacks of fructosamine assay include changes in serum level due to changes in albumin rather than blood glucose. According to one report, changes in albumin affect fructosamine levels significantly only if decreased albumin levels are due to increased catabolism (decreased half-life) or increased albumin loss, but not when there is decreased metabolism of protein. Reducing substances in serum may interfere with the assay in some methods.

December 20, 2009
Plasma (or Serum) Insulin Assay

Insulin was the first hormone measured successfully by radioisotope immunoassay, and insulin assay is now available in most sizable reference laboratories. Insulin is excreted primarily through the kidneys. In general, juvenile diabetics have low fasting insulin levels, and an OGTT using insulin determinations usually produces a flat curve. Mild diabetics have normal fasting insulin levels and display an insulin GTT curve that has a delayed rise, either to normal height or to a point moderately above normal; in either case the curve thereafter falls in a normal fashion. Decreased tolerance due to many other causes produces similar curves; an insulin OGTT has not been more efficient in uncovering subclinical diabetes than blood glucose OGTT. Some maintain that the ratio of insulin values to glucose values obtained on the same specimen during the OGTT is more reliable than insulin values alone. At any rate, most investigators believe that, at present, plasma insulin levels should not be used for diagnosis of diabetes mellitus.

Plasma anticoagulated with ethylenediamine tetraacetic acid (EDTA) is reported to produce plasma insulin values equal to serum, but heparin is said to be associated with plasma insulin values greater than serum.

Patients being treated with insulin frequently develop antiinsulin antibodies after approximately 6 weeks. These antibodies interfere with insulin RIA measurement by competing with insulin antibodies used in the test. Whether values will be falsely increased or decreased depends on the method used. Endogenous antibodies do not interfere with tolerance tests, since the quantity of endogenous antibody remains unchanged throughout the test; only the baseline value is affected.

December 20, 2009
Evaluation of Protein-Calorie Nutritional Status

Various studies have shown that a significant degree of malnutrition is frequent in hospitalized persons, ranging from 25%-50% of patients (depending on whether the population screened was a general or specialty group). In one report, 97% of surgical patients had at least one abnormal result on tests for nutritional status.

Classification of protein-calorie malnutrition

Although protein or caloric deficiency exists in grades of severity, a classification of patients according to pathophysiology and laboratory abnormalities requires analysis of late-stage deprivation. At a late stage, three basic patient types have been described: kwashiorkor, marasmus, and a mixed picture.

Kwashiorkor results from protein deficiency without total calorie deficiency. This condition is produced by a diet with adequate calories that are obtained almost exclusively from carbohydrate. This may result from a nonhospital diet that is low in protein or may be seen in hospitalized patients who are maintained primarily on IV dextrose. Severe stress, major illness, or surgery results in greatly increased utilization of body protein and may rapidly lead to protein depletion if there is not adequate replacement. These patients externally seem to be of normal weight or even overweight and may have edema. Kwashiorkor involves depletion of primarily visceral (nonmuscle) protein.

Marasmus is produced by prolonged deficiency of both protein and carbohydrates. Examples are starvation due to inability to eat and anorexia nervosa. These patients lose both fat and muscle mass and appear emaciated. Marasmus involves loss of primarily somatic (fat and muscle) protein rather than visceral protein.

The mixed category combines various degrees of protein deprivation with various degrees of carbohydrate and total calorie deficiency. It may also result from end-state marasmus (when both somatic and visceral protein are consumed) or may occur in a patient with moderate marasmus who undergoes severe stress, thus accelerating visceral protein loss.

Besides classic end-state cases there are far greater numbers of patients with malnutrition of lesser severity. In general, the greater the degree of deficiency, the greater the chance of unwanted consequences. These include increased postoperative morbidity and mortality and certain defined complications such as increased tendency toward infection, poor wound healing, and extended hospitalization.

Tests useful in patients with malnutrition

Functional categories of tests (i.e., information that tests can supply) in protein-calorie malnutrition include the following:

1. Tests that screen patients for protein-calorie deficiency
2. Tests that assess degree of deficiency
3. Tests that differentiate the various types of deficiency
4. Tests used to guide therapy

Procedures or tests available

1. Anthropometric measurements: triceps skinfold thickness; midarm circumference
2. Calculation of undernourishment based on body height and weight (percent of ideal weight or percent of preillness weight)
3. Biochemical tests reflecting visceral (nonmuscle) protein status: serum albumin and serum transferrin (also, serum iron-binding capacity, retinol-binding protein, serum prealbumin)
4. Metabolic indices: creatinine-height index (somatic protein or muscle mass) and urinary nitrogen excretion (protein catabolism)
5. Tests of immune status: skin tests with various antigens, total lymphocyte count

Anthropometric measurements

These procedures are designed to estimate fat and muscle wasting, which reflects somatic protein depletion. Triceps skin fold measurement is performed with special calipers and measures fat reserves. Midarm circumference is used to estimate lean body mass. Patient measurements are compared with values in standard tables.

Weight deficiency (weight loss)

Weight deficiency may be calculated either as a percentage of preillness weight (current weight/preillness weight) or as a percentage of ideal weight (current weight/ideal weight). Ideal weight requires measurement of height and use of ideal weight tables. Percent weight loss after hospitalization is also useful. In all the various measurements, a 10% weight loss is considered suspicious for protein-calorie deficiency. If this occurred before admission, it probably developed over a relatively extended period of time (there is no standard time period, but at least 4 weeks has been suggested and seems reasonable) rather than over a short period of time, which is more likely to be a fluid problem. After hospitalization, there is no time limit if the weight loss is not due to diuretics, fluid removal, or some other obvious cause. Edema and obesity may produce error in nutritional assessment by these methods.

Tests for visceral protein status

Serum albumin. Albumin is the major force maintaining plasma oncotic pressure. It is synthesized by the liver from amino acids. Decreased serum albumin levels result from decreased production, either from defective synthesis because of liver cell damage, deficient intake of amino acids (absolute protein intake deficit); or from disease- or stress-induced catabolism of body protein, which increases the need for dietary protein without a corresponding increase in dietary protein intake (relative protein intake deficit). The serum albumin level is thus considered an indicator of visceral (nonmuscle) protein status. Other serum proteins that have been used for the same purpose include transferrin, prealbumin, and retinol-binding protein.

Serum albumin has a serum half-life of about 20 days and begins to decrease about 2 weeks after onset of protein depletion. Mild protein deficiency is said to correlate with albumin levels of 3.0-3.5 gm/100 ml (30-35 g/L); moderate deficiency, 2.1-3.0 gm/100 ml (21-30 g/L); and severe deficiency, less than 2.1 gm/100 ml (21 g/L). Other etiologies for albumin decrease besides deficient protein intake include disorders of liver synthesis (cirrhosis, severe acute liver disease), extravascular protein loss (nephrotic syndrome, acute or chronic protein-losing enteropathy, extensive burns), and hemodilution (congestive heart failure). Albumin decrease, whether within or below reference limits, is seen in many severe acute and chronic illnesses. The exact mechanism is frequently uncertain or is sometimes due to more than one cause. Overhydration and dehydration also may change apparent albumin levels.

Serum transferrin. Transferrin has a serum half-life of about 9 days and begins to decrease about 1 week after onset of protein depletion. However, chronic iron deficiency, therapy with estrogen or estrogen-containing contraceptives, and the same severe acute and chronic illnesses that decrease albumin levels tend to elevate serum transferrin levels and could mask early change due to nutritional depletion. Transferrin can be measured directly in a variety of ways, usually by immunologic (antitransferrin antibody) techniques, or can be estimated using serum total iron-binding capacity (TIBC). TIBC is easier and less expensive for most laboratories. The formula most commonly used is: transferrin = (0.8 Ч TIBC) – 43. Mild protein deficiency correlates with transferrin levels of 150-175 mg/100 ml (1.5-1.7 g/L); moderate deficiency, 100-150 mg/100 ml (1.0-1.5 g/L); and severe deficiency, less than 100 mg/100 ml (1.0 g/L).

Serum prealbumin. Prealbumin is a carrier protein for retinol-binding protein and for a small part of serum thyroxine. Its serum half-life is about 2 days. Its serum concentration decreases in many severe illnesses. It is measured by immunologic techniques, most commonly by radial immunodiffusion or immunonephelometry. Prealbumin levels begin to decrease within 48-72 hours in response to protein malnutrition. However, like albumin, it is decreased by severe liver disease or as a temporary short- or long-term result of many severe acute or chronic illnesses.

Retinol-binding protein. Retinol-binding protein is the specific binding protein for vitamin A. Its serum half-life is only about 10 hours. It begins to decrease within 48-72 hours after onset of protein malnutrition and otherwise behaves like prealbumin. However, in addition, retinol-binding protein may decrease when renal function decreases (as frequently occurs in severely ill persons).

Most investigators have accepted serum albumin as the most practical marker for visceral protein depletion. When serial measurements of visceral protein indices are necessary, transferrin may be substituted because it responds faster than albumin to change in nutrition status. Transferrin can also be used if albumin measurement is invalidated by therapeutic administration of albumin. Comparisons of serum albumin with other parameters of malnutrition have generally shown that serum albumin levels have the best single-test correlation with patient outcome.

Metabolic indices

Creatinine height index (CHI). This calculated value is thought to provide an estimate of lean body mass, based on the theory that urinary creatinine (UC) output is related to body muscle mass. Height is used to relate patient data to data of normal (“ideal”) persons. The creatinine height index (CHI) has the advantage that it relates to skeletal muscle mass (somatic protein) rather than to liver production of serum protein (visceral protein). In classic marasmus, the CHI is markedly decreased, whereas the serum albumin concentration may be either within reference range or close to it. Another advantage is that edema does not greatly affect the CHI, whereas it might affect arm circumference measurement. The major disadvantage is need for accurate 24-hour urine collection. Also, urine ketone bodies can interfere with creatinine assay. Data for ideal urine creatinine excretion are presented in Table 37-11.

Nitrogen balance estimates. Nitrogen balance (NB), as measured by urine urea nitrogen (UUN), may be helpful in assessing the quantitative and compositional adequacy of nutritional therapy. The formula most often used is:

NB = (Protein intake / 6.25) – (UUN + 4)

when nitrogen balance is in terms of net gain (+) or loss (–) in grams of nitrogen per day and both protein intake and urine urea output are in grams per day. Reference limits are +4 to –20 gm of nitrogen/day. Protein intake (in grams/day) is usually estimated but can be measured if feeding is entirely through nasogastric tube or hyperalimentation. If the patient has oral food intake, only the food actually eaten rather than food provided should be used in the calculation. The 4-gm correction factor is supposed to compensate for urine non-urea nitrogen loss additional to the urea nitrogen. If additional loss occurs from the GI tract, fistulas, and so forth, such loss must be estimated and incorporated into the correction factor. The 24-hour urine collection must be complete, because incomplete collection results in a falsely higher value.

Tests of immune status

Moderate or severe protein-calorie malnutrition of any type often results in depression of the body immune system. This is reflected in decreased immune response (especially, delayed hypersensitivity response) to various stimuli.

Total lymphocyte count. There is a rough correlation of total lymphocyte count with degree of malnutrition. The correlation is closest in kwashiorkor (visceral protein depletion). Total lymphocyte counts of 1,200-2,000/mm3 (1.2-2.0 x 109/L) are said to be associated with mild protein depletion; counts of 800-1,200/mm3 with moderate depletion, and counts of less than 800/mm3 with severe depletion. However, there is considerable overlap between immunologic impairment and nonimpairment with counts higher than 1,000/mm3. Values less than 1,000/mm3 (1.0 x 109/L) are generally considered evidence of definite significant immunologic impairment. Total lymphocyte count is easy to obtain (WBC count x percent lymphocytes in the peripheral smear WBC differential result). Most investigators have found less correlation with eventual patient outcome than with serum albumin levels. One reason is that various other conditions may cause a decrease in total lymphocytes. Best correlation of total lymphocyte count to patient well-being seems to be associated with infection and with cancer therapy. When both albumin and total lymphocyte counts are significantly decreased, there is some reinforcement of significance compared with abnormality in either test alone.

Skin tests. Skin test response to various intradermally injected delayed hypersensitivity antigens such as Candida, mumps, and streptokinase-streptodornase provides an in vivo method to evaluate immune response. Reactions are measured at 24 and 48 hours. Various studies have shown a substantial correlation between lack of skin test reactivity to multiple antigens and an increased incidence of sepsis or postoperative complications and mortality. There is some disagreement in the literature on the predictive value of skin tests versus serum albumin levels, with the majority opinion favoring albumin. The major drawbacks to skin testing are the time interval required and the necessity for good injection technique.

Current status of tests for protein-calorie malnutrition

Patient screening for protein-calorie malnutrition. Although various institutions have different protocols, the most common practice that is emerging is to determine the percent of weight loss and the serum albumin level, either one alone or in combination, and to take into consideration the type of illness or therapy involved. Total lymphocyte count also seems to be widely used as an adjunctive test. The CHI is helpful if marasmus is suspected; serum albumin levels could be misleading if albumin were the sole criterion for possible malnutrition.

Tests to assess degree of deficiency. Serum albumin level is the most widely used single test. The CHI is also widely employed if marasmus is present.

Nutritional Deficiency Syndromes and Screening Tests
Overall nutritional status
1. Percent weight loss (at least 10% nondiuretic loss)
Marasmus (somatic protein and fat deficit due to total calorie deficiency; somatic protein = skeletal muscle lean body mass)
1. Somatic protein estimation
Creatinine-height index
Midarm circumference
2. Fat
Triceps skin fold thickness
Kwashiorkor (visceral protein deficit due to protein intake deficiency; visceral protein = liver-produced protein, including plasma proteins)
1. Serum albumin (or transferrin, prealbumin, retinol-binding protein)
2. Total lymphocyte count
3. Cell-mediated immunity
Mixed kwashiorkor and marasmus (deficit in both protein intake and total calories)
1. Tests abnormal in both deficiency groups.

Anthropometric measurements, serum transferrin determination, and tests of immune function are available but seem to be ordered more in university or research centers.

Tests to differentiate categories of malnutrition. In classic kwashiorkor, anthropometric measurements and CHI values are relatively normal, whereas serum albumin levels and other tests of visceral protein status are decreased. In classic marasmus, anthropometric measurements and the CHI are decreased, whereas results of visceral protein adequacy tests may be normal. Although immune status test results are depressed in severe marasmus, over the broad spectrum of marasmus severity they are not as severely affected as in kwashiorkor. It must be emphasized that patients may have some combination of overall protein-calorie deficiency and of severe protein loss and therefore may not have clear-cut differential test patterns.

Tests to guide nutritional therapy. Serum albumin levels and total lymphocyte count are still the most commonly used parameters of therapeutic response. Because serum albumin has a relatively long half-life and changes relatively slowly in response to renourishment, and also because fluid shifts influence albumin levels, an increasing number of investigators use serum transferrin or prealbumin levels rather than albumin levels to monitor therapy. Some find that urinary nitrogen excretion data are very helpful in determining when a positive nitrogen balance has been achieved, but others do not believe that it is necessary.

December 16, 2009
Serum Magnesium Abnormalities

Magnesium is the fourth most common body cation (after sodium, potassium, and calcium) and the second most common intracellular cation (after potassium). About half is located in soft tissue and muscle cells and about half is in bone. Only 1%-5% is extracellular. Most body magnesium is derived from food intake. About one third of dietary magnesium is absorbed, with the absorption site being the small intestine. Body magnesium is excreted by the kidney, primarily through glomerular filtration. Some tubular reabsorption also takes place. About 33% of serum magnesium (literature range, 15%-45%) is bound to serum proteins, 15% is complexed, and about 50% is free in the ionized form. Of the protein-bound fraction, about 75% is attached to albumin and most of the remainder to alpha-1 and alpha-2 globulin. Albumin thus carries about 30% (range, 25%-33%) of total serum magnesium. PTH is a very important regulator of magnesium blood levels through regulation of renal tubule reabsorption.

Magnesium is important in protein synthesis, enzyme activation, and oxidative phosphorylation. It influences renal exchange of potassium and hydrogen ions and affects calcium levels. It also has an important role in nervous system control of muscle at the neuromuscular junction, where it slows neuromuscular impulse transmission by inhibiting acetylcholine. The major clinical symptoms of magnesium disorders are neuromuscular. Magnesium deficiency enhances muscle fiber excitability due to increased activity of acetyl-choline; this is manifested by muscle tremor, which can progress to seizures and tetany. Mental abnormalities include confusion, anxiety, and hallucination. Magnesium excess conversely displays antagonism of nerve impulse transmission and results in muscle weakness. Magnesium also exerts some effect on heart muscle. Decreased magnesium levels may produce or aggravate cardiac arrhythmias, whereas toxic levels of magnesium may be associated with heart block. Hypomagnesemia also potentiates the toxic effects of digitalis.

Magnesium deficiency. Magnesium deficiency has been reported in about 10% (range, 7%-11%) of hospitalized patients. Some of the etiologies of hypomagnesemia are listed in the box. In addition, it has been reported that hypomagnesemia frequently accompanies hyponatremia (22%-27% of hyponatremic patients); hypocalcemia (22%-32% of patients); and hypophosphatemia (25%-29% of patients). Several studies also found that hypomagnesemia is frequent in patients with hypokalemia (38%-42%), but one study reported only 7%. Postoperative patients on IV feeding are reported to have a short-term, temporary 20% decrease in serum magnesium levels. Similar findings have been reported 12-24 hours after acute myocardial infarction, returning to previous levels by 48 hours, but not all studies agree.

Excess magnesium. Increased serum magnesium levels are most often due to oliguric renal failure, which prevents excretion of magnesium. Overuse of magnesium compounds is an occasional etiology.

Laboratory tests. RBCs contain 2-3 times the concentration of magnesium found in serum. Artifactual hemolysis thus may produce a significant increase in assay values. Skeletal muscle contains about 10 times the serum concentration. Since about 30% of serum magnesium is bound to albumin, and assays measure total magnesium levels, hypoalbuminemia will artifactually decrease serum magnesium levels.

Magnesium Disorders
Magnesium deficiency
Alcoholism
Malabsorption
Malnutrition
IV fluids without magnesium
Severe diarrhea
Diabetic ketoacidosis
Hemodialysis
Hypercalcemia
Congestive heart failure
Artifact (hypoalbuminemia)
Certain medications
Loop and thiazide diuretics
Cyclosporine
Cisplatin
Gentamicin
Magnesium excess
Oliguric renal failure
Overuse of magnesium-containing compounds
Artifactual (specimen hemolysis)

Various reports emphasize that serum magnesium values may not always truly reflect total body magnesium levels, since serum values may be falsely elevated in dehydration and falsely decreased in hemodilution with or without clinical edema or hypoalbuminemia. However, this problem is not unique to magnesium.

December 15, 2009
Serum Parathyroid Hormone-Related Protein (PTHrP)

Since many patients (50% or more) with cancer and hypercalcemia do not have demonstrable bone metastases or PHPT, it has long been suspected that the cancer could be producing a parathyroid hormonelike substance. The parathyroid hormone-related protein (PTHrP) molecule has a C-terminal end and an N-terminal end like PTH; in addition, a portion of the PTHrP amino acid sequence is identical to that of PTH, although the majority of the PTHrP molecule is not. Also, it has been found that certain normal tissues can produce PTHrP (including the keratinized layer of skin epidermis, lactating breast tissue, placenta, adrenal, and a few others). PTHrP has recently been isolated and cloned, and antibodies have been obtained that react against it. Several investigators have reported results using homemade test kits, and one commercial kit is now available. Results thus far with these first-generation kits show that about 50% (range, 20%-91%) of patients with solid malignancies and hypercalcemia have increased PTHrP levels. Another 20% have bone metastases that could account for hypercalcemia without hormonal basis. It is currently thought that the other 30% may be producing some type of altered PTHrP that is not being detected by current antibodies. PTHrP assay may be useful when PTH assays fail to give expected results in patients with malignancy or give results that are borderline or slightly overlapping in nomogram areas between PHPT and tumor patients. However, PTHrP assays are not all alike and it is necessary to find a laboratory or kit that gives superior results.

December 15, 2009