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4
BIOLOGIC MAKERS OF EFFECT
Biologic markers of effect are perhaps the most important set of markers. Markers of effect permit the early identification of adverse effects of toxic xenobiotic exposure. They constitute the missing link in the continuum of disease development, linking markers of exposure and susceptibility with onset of disease and offering the possibility of detecting disease at the very early stages of development. They offer the most potential for clinical intervention before irreversible effects have occurred. With proper use of such markers, carcinogenesis can be detected before clinically apparent tumors result, and nephrotoxicity can be identified while sufficient kidney function still remains.
This chapter focuses on relatively noninvasive measurements of early genitourinary consequences of human exposure to noxious chemical, biologic, or physical agents. It discusses available markers of renal effects, their limitations, and directions for future work, but it excludes in vitro procedures extensively used for evaluating cytotoxic effects in animals (which is covered in Chapter 5) or for studying mechanisms of nephrotoxicity. It also discusses markers of carcinogenesis that signal tumor development and address other disorders, such as interstitial cystitis.
Our objective here is to identify and evaluate markers of general or specific effects on renal function and integrity. Measurement of these markers is preferably minimally invasive, accurate, and suitable for screening populations at risk. A number of techniques have been developed to assess renal function, but these techniques generally lack the sensitivity and specificity necessary for detecting preclinical disease or are not applicable to large populations. For example, renal biopsy and radioisotopic procedures are not appropriate for screening, and classical clearance procedures are seldom reproducible to within ±10%, even in the laboratory. Compromises have therefore been made with respect to convenience, sensitivity, and invasiveness. The conventional markers are reviewed from this perspective, and newer results from cellu
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lar and molecular studies are presented in light of their potential to provide new markers of effect that will be more sensitive to early nephrotoxicity and can be applied widely to at-risk populations. The conventional markers also can play an important role in validating new markers, particularly in people with compromised renal function who are exposed to additional potentially damaging events. The loss of a small number of tubular cells in a person with only minimal residual kidney function can lead to large changes in conventional markers whereas the same loss in a normal person would be undetectable. Such a change, now easily detectable in a compromised person, can be used to validate new markers against conventional markers.
EXTERNAL VISUALIZATION
Several techniques permit external visualization of the kidney and delineation of functional changes. Radiopaque contrast media and ultrasonic agents permit imaging of changes in blood or urine flow or in renal solute accumulation and secretion. Similar results are routinely obtained with gamma-emitting radioisotopes. In addition to the risks associated with radiation exposure, these tests do not reflect subtle changes in renal function and are therefore limited in their applicability to population screening.
A visualization technique that might hold promise and that has been applied to small animals is magnetic resonance imaging (MRI). Acara et al. (1991) observed conspicuously hyperintense regions in renal papillae in rats and related them to early hydronephrotic changes. Further improvements in MRI might make the technique more useful for screening human populations, but it is still time-consuming and expensive.
URINARY CLEARANCE MEASUREMENTS
Direct
Measurement of urinary clearances is not convenient for use in large populations at risk, but it continues to constitute a basic approach to evaluating renal function. Indeed, the introduction of the clearance concept into renal physiology by Moller et al. in 1929 provided for the first time a relatively simple and informative measure of renal function and was a critical contribution to the study of the kidney. Urinary clearance (C) is the virtual volume of plasma that is cleared per unit time by excretion into urine; it is expressed as Cx = Ux V/Px, where Ux V is the amount of substance x excreted per unit time and Px is the plasma concentration of substance x. Depending on the solute chosen, clearances can provide information on glomerular and tubular function; they are useful under many conditions for evaluating functional integrity of the kidney in humans or animals.
The direct measurement of clearance, however, has disadvantages, especially for the study of large populations. It is clear from the formula Cx = Ux V/Px that it is essential to keep Px constant during the clearance period or at least to be able
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to calculate the mean plasma concentration of the chosen solute. For exogenous solutes, such as inulin (often used to evaluate renal function), this requirement is best met by continuous intravenous infusion of I hour or longer, but it can at times be approximated by subcutaneous or intramuscular depot injection. In either case, blood has to be sampled repeatedly to ensure the required constancy of P. The procedure is simplified by reliance on an endogenous solute, such as creatinine, which is usually produced in the body at a constant rate and normally excreted almost entirely by glomerular filtration. Creatinine clearance can be a measure of glomerular filtration rate. Clearance of endogenous urea has also been used to evaluate glomerular function, but it suffers from the double disadvantage of less-constant plasma concentration than that of creatinine and substantial influence by urinary flow rate.
The need for accurately timed and complete urine collection presents a second difficulty in clearance procedures. Because of the cumulative errors arising from lack of constant plasma concentrations and from inaccurate timing or incomplete urine collection (and because of analytic errors), clearance determinations are seldom reproducible to within ±10%, even in a laboratory setting. Such low reproducibility might be adequate for many clinical purposes, but it interferes with attempts to demonstrate early changes in the ability of the kidney to excrete a given solute. In addition, early effects of nephrotoxicants might result in diminution of glomerular reserve capacity without changes in overall clearance (as described in Chapter 2). The glomerular reserve capacity reflects the ability to increase the glomerular filtration rate in response to stimuli. A failure to elicit such a response implies the presence of a subclinical disease process.
Total functional reserve capacity of the kidney can be measured by, for example, imposing a stress, such as ingestion of a high-protein meal. The increase in creatinine clearance during pregnancy can also reflect functional reserve capacity. For tubular secretion or reabsorption, total capacity is evaluated at saturating levels of a test solute, in which case secretion is expressed as excreted load minus filtered load, whereas reabsorption is calculated as filtered load minus excreted load. These techniques require determination of the glomerular filtration rate (GFR) as well as prolonged infusion of large amounts of solute and therefore are not suitable for routine screening.
Direct clearance measurement takes place primarily in the laboratory or special clinic. It can be simplified in some cases by use of radioisotopes, but this is not appropriate for general screening. It would be useful if convenient and sensitive chemical procedures could be developed for such compounds as chromium ethylenediaminetetraacetic acid (Cr-EDTA) and iodothalamate, both good solutes for measurement of GFR but so far used only in the radiolabeled form.
Indirect
A number of indirect measures of
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renal clearance have been proposed. They are generally simple but lack sensitivity and precision, as can be illustrated by reference to the formerly extensively used measurement of the rate of excretion of a chemical after injection of a standard dose of some substance. For instance, the dye phenol red (PSP) is excreted by tubular secretion, and the rate of its recovery in urine is therefore controlled by independent variables, such as renal plasma flow, the integrity of tubular transport mechanisms, and a linear dependence of the rate of transport on plasma concentration of the dye at the dose used. Measurement of fractional excretion of a standard dose of PSP over 15 or 30 min can yield little information on specific aspects of renal function.
A more commonly used indirect method measures the declining slope of plasma concentration of an injected solute after a suitable equilibration period. An example is the estimation of GFR with iodothalamate.
Another indirect measurement of urinary clearance is based on the assumption that creatinine is filtered and excreted at a constant rate. Even in the presence of diurnal variations in GFR, normalizing urinary concentrations of a specified substance by the urinary concentration of creatinine permits conclusions to be drawn from spot urine samples and avoids the difficulty of obtaining 24-hour collections. Any deviation of a solute-to-creatinine concentration ratio from normal is equated to a corresponding change in the clearance of this solute. Normalization can also be achieved on the basis of total urine concentration as reflected in its specific gravity, with the empirically derived mean value of 1.018 as a reference point. Because GFR undergoes diurnal variation, spot samples should be collected at the same time each day. It must be kept in mind that specific lesions cannot always be blamed for a change in fractional excretion. For instance, the fractional excretion of filtered sodium normally falls below 1%. If GFR becomes depressed for any reason, maintenance of normal sodium excretion in the face of a reduced filtered load necessitates an increased fractional excretion.
In selected cases, clearance values can be computed indirectly from analysis of serum. The solutes most commonly studied in this manner are creatinine and urea. Creatinine is synthesized and excreted at an approximately constant rate. It follows that its steady-state concentration in plasma will vary indirectly with GFR. The agreement between measured creatinine clearance and that predicted from plasma creatinine concentration shows considerable variation. The predicted creatinine clearance is based on the formula Ccr = [(140-age in years) (weight in kg)]/72Scr' where C is in milliliters per minute and S in milligrams per deciliter (Cockroft and Gault, 1976), for men. For women, 85% of the value is chosen. Both may be expressed in terms of body weight or body surface area. Blood urea has been similarly used as an indicator of glomerular function, but variations in the rate of urea synthesis and tubular reabsorption render it unsuitable for quantitative
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purposes. Attempts have also been made to use plasma concentrations of beta2-microglobulin to measure GFR; this is further considered in the next section. All these methods are of qualitative, rather than quantitative, use.
URINALYSIS
Toxic injury to the kidney can involve any of the tissue compartments of the urinary tract. Ultimately, the products of such injury—whether sloughed necrotic renal epithelial cells themselves or the products of the functional, inflammatory, and reparative responses that the injury provokes—find their way into the urine. Simple urinalysis remains perhaps the most useful screening test for the detection of renal injury in large populations. Changes in urinary excretion of numerous solutes have been suggested as markers of exposure and effect. Bowers et al. (1992), for instance, showed that urinary porphyrin can serve as a marker of mercury accumulation and nephrotoxicity. It is unlikely, however, that porphyrin analysis provides more information than some of the simpler tests routinely performed.
Total Urinary Solute Concentration
In almost all cases of nephrotoxic injury, an early and reliable marker is the failure of the kidney to excrete concentrated urine. The failure is caused by the inability of the damaged kidney to generate and maintain a high osmolar concentration in the renal papilla. The two routine tests of this capacity are the urinary specific gravity (SG) and osmolality.
Urinary SG correlates well with the osmolar concentration with some exceptions. Normally, urinary SG exceeds 1.020 after overnight water deprivation, and values below 1.015 indicate a concentration defect. Care must be taken to correct the determined values for abnormally high glucose or protein concentrations. SG can be determined directly with a hydrometer. Indirect, easy-to-use measurements are gaining acceptance. The most common of these is based on the change in ionization of a polyelectrolyte as a function of the ionic strength of urine; the resulting change in pH is noted with a dipstick.
Osmolality can be measured with vapor-pressure osmometry or freezing-point depression. In the United States, subjects ingesting a normal diet excrete urine of up to around 900–1,200 mOsmol/L. By comparison, the osmolality of plasma is 280 mOsmol/L. In chronic renal disease or after acute toxic injury, the ability to concentrate urine maximally is impaired; and with severe injury, the maximal urine osmolality approaches that of plasma.
Dipsticks
Dipsticks have found extensive use for evaluation of urinary pH and the presence of proteins, glucose, hemoglobin, and bile pigments.
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pH. This is not greatly affected by most renal diseases, and the ability to acidify urine maximally is preserved even in severe forms of renal dysfunction. However, some forms of renal injury are associated with defects in acid excretion. Such so-called tubular acidosis can be induced by some xenobiotics, by solvents and metals, and by obstruction of the urinary tract. Urine excreted in cases of lead, cadmium, and toluene toxicity might be alkaline. The samples should be collected after overnight water deprivation to avoid the alkaline pH produced by stimulation of gastric HCl production.
Proteinuria (discussed in detail later). The colorimetric reagent strip of most dipsticks uses tetrabromphenol blue for the semiquantitative detection of protein. The test is more sensitive for albumin than for other urinary proteins, and its sensitivity is greatly diminished at high pH.
Glucosuria. Like proteinuria, glucosuria generally accompanies acute renal injury after exposure to nephrotoxicants. The glucose oxidase reagent is highly specific for glucose.
Hematuria. Presence of hemoglobin (or large amounts of myoglobin) is readily demonstrated. The presence of red blood cells in the urine is abnormal. It does not necessarily reflect renal lesions themselves, inasmuch as diseases in any portion of the urinary tract can lead to this finding.
Bile pigments. Nephrotoxicants often damage liver and red-cell membranes, so bilirubin and urobilinogen are important indicators of liver injury. Because bilirubin is unstable and light-sensitive, samples should be stored in the dark.
Microscopic Analysis
Little information is usually gained from microscopic examination of urinary sediment if chemical analysis had negative results. In large screening studies, microscopic examination of urine should be reserved for urine samples whose chemical screens were positive. First morning specimens, which are highly concentrated, as opposed to those passed later in the day, are not useful for this purpose, because cells rapidly deteriorate in hypertonic bladder urine. Therefore, freshly voided samples should be used only after partial hydration, which will result in a lower urine specific gravity. Preservatives can be added to ensure the integrity of cellular elements. This preservation will be especially important when more sophisticated techniques—such as immunocytology, in situ hybridization, and polymerase chain reaction (PCR), are introduced to examine these specimens (see the next major section, on markers of cytotoxicity). Formaldehyde-releasing tablets are now available as a preservative, but all preservatives might interfere with routine tests. For example, false-positive results for protein and glucose have been observed after addition of the preservatives thymol and formalin, respectively.
Preparation of the sediment should be standardized. Most commonly, 15 ml of urine is spun at 2000 g for 5 min. The supernatant is partially decanted and the
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sediment resuspended in 1 ml of supernatant. In such a preparation, the normal concentration of red cells and white cells is 1-2 cells per high-power field. The presence of casts, which can be formed especially at high salt concentrations in acidic urine, reflects injury to the glomerular capillary wall (in the case of red-cell casts) or tubules; the presence of granular and hyaline casts probably depends on the combined effects of tubular epithelial damage, inflammatory reactions, and abnormal protein excretion and aggregation.
Study of urinary cells is discussed in greater detail in the next major section. Normal urine specimens can contain epithelial cells and cells of the pelvic, ureteral, bladder, and urethral epithelium that cannot be distinguished by routine light microscopy. Some inflammatory cells can also found. A recent study by Mandal (1986) failed to identify tubule cells in normal urine with electron microscopy. Under pathologic conditions, cell identification requires a variety of techniques to identify phenotypic markers. Granular casts, consisting of contents of disintegrated cells, and Tamm-Horsfall protein (see the next section) are commonly found in the urine from patients with acute renal failure (ARF), and acute or chronic tubulointerstitial disease. Muddy-brown casts, coarsely granular casts, red cells and casts, and white cells are all associated with ARF. Their presence correlates with the degree of damage, so mild cytotoxicity is accompanied by few urinary abnormalities. However, major renal impairment can be present in acute interstitial nephritis with little or no abnormality of the urinary sediment. Acute interstitial nephritis with prominent peripheral eosinophilia can lead to the presence of eosinophils in urine.
Proteinuria
Under normal circumstances, the glomerular capillary wall provides a barrier to the filtration of protein. Permeation of the glomerular basement membrane by proteins is influenced by the size, charge, and rigidity of individual protein molecules. Small uncharged molecules, such as horseradish peroxidase, pass freely into the urinary space. Negatively charged molecules of intermediate size, such as albumin, are impeded at the endothelial-cell surface and the proximal layers of the glomerular basement membrane. Uncharged or positively charged molecules, such as myeloperoxidase, permeate the glomerular basement membrane and are retarded at the slit diaphragm of epithelial cells. The net effect of those properties is to exclude relatively large, negatively charged, and rigid proteins from passage into the urinary space. Thus, although albumin and gamma globulin account for the preponderance of serum protein in normal humans (5.0 and 1.5 g/dL, respectively), less than 60 mg and 20 mg, respectively, is excreted in the urine daily. Serum proteins that weigh less than 20,000 daltons (low-molecular-weight proteins) and peptide hormones contained in plasma pass readily into the urinary space, but almost the entire
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filtered load is later reclaimed in the proximal tubule.
Measurement of Urinary Protein
The urinary dipstick is the most common device for semiquantitative measurement of urinary protein. Although it can detect protein concentrations as low as about 10–15 mg/dL, it is relatively insensitive and unreliable at concentrations below 30 mg/dL, yielding negative or trace-positive results in more than half such samples tested (Rennie and Keen, 1967). Although the test is sensitive to small quantities of negatively charged proteins, such as albumin, it is insensitive to positively charged proteins, such as some immunoglobulin light chains, and to Tamm-Horsfall protein. Low-molecular-weight proteins (which are less negatively charged than albumin), even when present in abnormal amounts, do not usually reach a concentration high enough to give a clearly positive test result.
Urinary protein can also be estimated with precipitation. Urinary protein is precipitated either by adding 5% sulfo-salicylic acid, 10% trichloroacetic acid, or concentrated nitric acid to the urine or by heating the urine and adding 5% glacial acetic acid. Those methods detect positively charged proteins, as well as albumin, but they have the same limitations as the dipstick test in quantifying total protein; that is, small amounts of protein might not be detected in dilute urine, and normal amounts of protein might be detected in concentrated urine. For mass screening purposes, precipitation tests offer little advantage over the dipstick method and are more difficult to perform. Neither dipstick tests nor precipitation tests are useful in testing for abnormal low-molecular-weight proteinuria.
Neither the dipstick methods now in use nor the usual qualitative tests for acid precipitation of protein are sensitive enough to function as reliable screening tests for the detection of borderline or low concentrations of proteinuria (200–500 mg/g of creatinine) in random urine samples. For example, the sample sensitivity of two dipstick methods was less than 50% in consecutively acquired specimens, and the negative predictive value, or the ability to establish the absence of proteinuria, was only 64–69% (Allen et al., 1991). Quantitative assessment of protein excretion with measurement of the protein-to-creatinine ratio has been recommended to supplement the dipstick in screening for proteinuria in cases in which misclassification would lead to serious problems (Shaw et al., 1983). Interpretation of borderline positive results for protein in screening examinations is further clouded by the fact that functional proteinuria—elicited by upright posture, by exercise, and even by excitement—is frequently encountered, particularly in adolescents and young adults (Houser, 1987; Houser et al., 1986). Woolhandler et al. (1989) thought that screening healthy, asymptomatic young adults for proteinuria was not helpful, in that population-based studies showed that less than 1.5% of patients with a positive result for protein
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on the dipstick test had a serious and treatable disorder of the urinary tract. However, the yield might well be much higher in selected populations, such as hospitalized patients, older subjects, or those exposed to environmental hazards.
Albuminuria
Albumin is a 69-kilodalton polyanionic macromolecule with a Stokes radius of 3.6 nm, and a pI of 4.8. In urine, it is considered a high-molecular-weight protein. Albumin is synthesized in the liver and has a relatively high concentration in the plasma (about 4 g/dL). Because this high concentration of albumin is maintained within the vascular space, it generates so-called ''oncotic pressure'', which prevents fluid from moving into the tissues and causing edema. Albumin is thought to exist almost entirely in monomeric form in biologic fluids. Some albumin is normally filtered by the glomeruli with low-molecular-weight proteins. Filtered protein is primarily reabsorbed in the proximal tubules by brush-border enzymes. Albumin has less affinity for the brush-border enzymes than does the low-molecular-weight protein beta 2-microglobulin. Absorption of beta2-microglobulin averages 99.7%, but that of albumin only 90%. In normal people, the fractional excretion of albumin is about 90 times higher than that of beta2-microglobulin (Peterson et al., 1969). Isolated albuminuria without increased excretion of low-molecular-weight proteins results from change in the glomerular filtration of high-molecular-weight proteins and thus has been termed glomerular proteinuria.
Various mechanisms have been proposed to explain increases in glomerular filtration of albumin. In advanced renal disease, when GFR is 25–30 mL/min, the remaining functional nephrons might have increased perfusion, which could disrupt the cellular integrity of capillary membranes and lead to increased albuminuria (Hostetter et al., 1982). Reduction in the fixed anionic charge of the glomerular filtration barrier enables greater leakage of serum albumin across the glomerular basement membrane. Nephrotic syndrome induced by puromycin aminonucleoside is associated with reduction of glomerular anionic sites (Michael et al., 1970). Conversely, if the serum protein itself is modified to be less negatively charged, it might result in increased filtration, as has been proposed for glycosylated albumin in diabetes (Ghiggeri et al., 1985). In general, then, the clinical finding of isolated urinary excretion of high-molecular-weight proteins, particularly albumin, is seen as a manifestation of disturbances of the glomerular filtration barrier.
Increased excretion of albumin is usually the result of glomerular injury but sometimes is the consequence of reversible hemodynamic changes. Exercise, fever, infusions of epinephrine or norepinephrine, emotional stress, prolonged assumption of the lordotic position, and congestive heart failure are often accompanied by mild to moderate albuminuria. Those stresses amplify the excretion of albumin when albuminuria is already present.
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Proteinuria in excess of 1 g/g of creatinine, or 1 g/day in an adult, is almost always indicative of glomerular injury. Excretion of large amounts of abnormal, positively charged immunoglobulin light chains, as in multiple myeloma, constitutes the chief exception to this rule. Although, with the foregoing exception, albumin is always the predominant constituent, proteins of higher molecular weight are excreted in small amounts in proportion to the degree of injury to the glomerular barrier. Thus, in minimal-change childhood nephrosis, in which glomeruli appear normal with light microscopy, virtually no proteins larger than albumin appear in urine, but in inflammatory or infiltrative glomerular diseases, the proteinuria is "nonselective," so urine contains variable amounts of high-molecular-weight plasma proteins, such as immunoglobulin G.
Day-to-day variation of 35–50% in urinary albumin excretion is well documented (Giampetro and Clerico, 1990). Normalizing urinary albumin with creatinine (discussed earlier) can increase the sensitivity and specificity of the test, but it requires a second assay, which increases cost and imprecision. Besides the physiologic factors noted above, various disease states and a number of nephrotoxic agents can increase albuminuria as well.
Microalbuminuria
The standard detectability limits for screening albuminuria are far above the normal range; this decreases the rate of false-positive results and thereby approaches a specificity of 100% but substantially reduces the sensitivity (Steffes et al., 1989). The hallmark of overt diabetic nephropathy is dipstick-positive proteinuria, as seen with "Albustix" (Ames Company). However, by the time the specified albuminuria (250–300 mg/L of urine or 250–300 mg/24 hours) is present, the progression to end-stage diabetic renal failure might be inexorable. In the patient with Type I or insulin-dependent diabetes mellitus (IDDM), once persistent proteinuria of this degree develops, the progression to ESRD is predictable (Konen et al., 1990; Mathiesen et al., 1984). Before clinical (dipstick-positive) proteinuria appears, there is a period of increased albumin excretion not detectable by dipstick (Mogensen, 1987). The small amount of albumin excreted can be conveniently detected with radioimmune assays. This "microalbuminuria" is said by some to be a marker of the likelihood of diabetic nephropathy. However, its prognostic utility in individual cases is uncertain, in that many patients with diabetes and microalbuminuria do not develop dipstick-positive proteinuria even after many years. Microalbuminuria in the absence of hypertension and decreased creatinine clearance does not accurately elucidate the severity of the underlying glomerular lesion in patients with type I diabetes (Chavers et al., 1989).
There has been disagreement regarding the albumin excretion that constitutes microalbuminuria. In 1985, a consensus conference agreed to define microalbuminuria as a urinary excretion
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rate (UAER) of greater than 20 μg/min but no higher than to 200 μg/min in an overnight or 24-hour sample (Mogensen et al., 1985–1986). A UAER of 20–200 μg/min is approximately equivalent to 30–300 mg/24 hr or 3–30 mg/mmol creatinine.
Low-Molecular-Weight Proteinuria
Virtually all filtered low-molecular-weight proteins are reabsorbed in convoluted and straight portions of the proximal tubule. Brush-border receptors have a considerably higher affinity for low-molecular-weight proteins than for albumin (which is why the concentration of these proteins in urine is so low), and a high absorptive capacity exists in relation to the normal filtered load for all low-molecular-weight proteins that have been tested (Maack et al., 1985). Nevertheless, with an increase in filtered load, urinary excretion generally increases well before the tubular maximum for absorption is reached (Waldmann et al., 1972).
Because low-molecular-weight proteins are reabsorbed solely and virtually completely in the proximal tubule, their appearance in urine in increased amounts is generally taken to indicate a reduction in proximal tubular function and an early sign of proximal tubular damage. But low-molecular-weight proteinuria can have other causes that lead to increases in the plasma concentration and glomerular filtration of the proteins. An increase in beta2-microglobulin occurs in lymphoproliferative malignancy. An increase in the plasma concentration can also be secondary to renal insufficiency; functional competition for absorption at brush-border sites by charged molecules, such as lysine, arginine, and aminoglycosides; and nonrenal febrile diseases.
One of the important low-molecular-weight proteins in urine is beta 2-microglobulin, whose molecular weight is 11800. For accurate assay results, urine must be made alkaline to prevent hydrolysis of the protein. The protein was isolated in 1968 by Beggard and Bearn from the urine of patients with two conditions characterized primarily by proximal tubular damage: Wilson's disease and chronic cadmium poisoning (Beggard and Bearn, 1968). Urinary beta2-microglobulin has been used often to detect proximal tubular injury in clinical and experimental settings and to test for toxic effects of environmental exposures and antibiotic and chemotherapeutic agents.
Normal adults produce 150–200 mg of beta2-microglobulin a day. It is cleared from the plasma almost completely by the kidney with a half-life of about 2 hours in persons with normal renal function. Serum concentration averages 2.0 mg/ml (range, 1.1–2.7 mg/ml), and the normal urinary excretion is less than 370 μg per day (Schardijn and Statius van Eps, 1987). An increase in beta2-microglobulin production occurs whenever nucleated-cell turnover increases and results, even in the absence of renal malfunction, in raised serum concentration and urinary excretion. When serum beta2-microglobulin exceeds 5 mg/ml, its urinary excretion is invariably high,
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Marker
References
Growth factors
e-cadherin
Bringuier et al., 1993
EGF and EGF receptor
Fuse et al., 1992, Harney et al., 1991 a,b; Ishikura et al., 1991; Kageyama et al., 1991; Messing et al., 1987; Neal et al., 1989, 1990; Rao et al., 1993; Smith et al., 1989; Theodorescu et al., 1991; Wood et al., 1992
fgf
Ravery et al., 1992; Valles et al., 1990
P-glyprotein
Kageyama et al., 1991; Moriyama et al., 1991a
Plasminogen activator
Hasui et al., 1992; Hiti et al., 1990; See, 1992
tgf-beta and tgf-alpha
Hiti et al., 1990; Kawamata et al., 1992
tnf
Hitti et al., 1990
Differentiation
amf
Javadpour and Guirguis, 1992; Nabi et al., 1992
Cytokeratin
Basta et al., 1988; el-Mohamady et al., 1991; Helmy et al., 1991; Konchuba et al., 1992; Schaafsma et al., 1990; Sumi et al., 1990
ECP
Lose and Frandsen, 1989
EMA
Ring et al., 1990
F-actin
Rao et al., 1991, 1993
G-actin
Rao et al., 1993
PNA, WGA
Orntoft et al., 1988
Blood-group antigens
ABH
Das and Glashan, 1988; Das et al., 1986; Limas, 1990; Malmstrom et al., 1991; Orntoft et al., 1988; Sanders et al., 1991; Tichy et al., 1991; Yamada et al., 1991
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Marker
References
Due ABC 3
Decken et al., 1992
HLA
Cordon-Cardo et al., 1991; Eryigit and Kirkali, 1990; Levin et al., 1991, 1992; Nouri et al., 1990; Tomita et al., 1990
Lewis-X
Limas, 1991; Matsusako et al., 1991
Lewis antigens
Fradet et al., 1990a; Langkilde et al., 1991a,b; Limas, 1991; Matsusako et al., 1991
Tumor-associated antigens
10D1
Hijazi et al., 1989
12F6
Hijazi et al., 1989
19A211
Cordon-Cardo et al., 1992; Fradet et al., 1990a
2A6
Messing et al., 1984
2E1
Messing et al., 1984
3-48-2, 48-1, 3-50-3
Summerhayes et al., 1985
3-71-1, 94-3
Summerhayes et al., 1985
3C6
Summerhayes et al., 1985
3G2-C6
Lin et al., 1988; Young et al., 1985
4-72-2
Summerhayes et al., 1985
486P 3/12
Arndt et al., 1987; Huland et al., 1990, 1991
6D1
Hijazi et al., 1989
7C12
Hijazi et al., 1989
8-30-3, 771-, 2-94-2
Summerhayes et al., 1985
9A7
Messing et al., 1984
AN43
Liebert et al., 1989
BB369
Liebert et al., 1989
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Marker
References
BIUH4, BIUH6, BIUH9
Guo, 1992
BL 2-10D1
Longin et al., 1989
BLCA-8
Lose and Frandsen, 1989; Walker et al., 1989
C3
Young et al., 1985
CA50
Morote et al., 1990
CEA
Boileau et al., 1987; Morote et al., 1990; Piana et al., 1991
DD23
Bonner et al., 1995; Grossman et al., 1992
G4,E7
Chopin et al., 1985
GF 26.7.3
Baricordi et al., 1985
HBA4, HBE3
Masuko et al., 1984
HBE10
Masuko et al., 1984
HBF2
Masuko et al., 1984
HBG9
Masuko et al., 1984
HBH8
Masuko et al., 1984
J143
Fradet et al., 1984, 1986
M344
Bonner et al., 1993; Cordon-Cardo et al., 1992; Fradet et al., 1987; Rao et al., 1993
Mano 4/4
Arndt et al., 1987
OM5
Fradet et al., 1984, 1986, 1990b
P7 A 5-4
Ben-Aissa et al., 1985
RBS-31, RBS-85, RBA-1, HBP-1
Masuko et al., 1989
SK 4H-12
Ben-Aissa et al., 1985
T16
Bretton et al., 1989; Fradet et al., 1984, 1986, 1990b
T23
Fradet et al., 1984, 1986
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Marker
References
T43
Fradet et al., 1984, 1986, 1990b
T87
Fradet et al., 1984, 1986
T110
Fradet et al., 1984, 1986
T138
Fradet et al., 1984, 1986, 1990b
Thomsen-Friedenrich
Yamada et al., 1991
Tu-MARK-BTA
Yogi et al., 1991
urine TPA
Carbin et al., 1989; Morote et al., 1990
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TABLE 4-5 Potential Biologic Markers of Prostatic Cancer
Marker
References
Clinical and pathologic markers
Atypical adenomatous hyperplasia
Bostwick et al., 1993
Hyperplasia metaplasia atrophy
Morote et al., 1986
Host inflammatory response
van Weerden et al., 1993
Morphologic variants of cancer
Davies et al., 1988; Lloyd et al., 1992; Partin et al., 1989
Prostatic intraepithelial neoplasia
Montironi et al., 1990
Tumor grade, (Gleason grade, nuclear grade)
Abrahamsson et al., 1987
Tumor stage
Azzopardi and Evans, 1971; Thompson, 1990
Tumor volume
Helpap, 1988
Morphometric markers
Chromatin abnormalities
Shah et al., 1987
Nuclear abnormalities
Shah et al., 1987; Umbas et al., 1992
Nuclear abnormalities
Wenk et al., 1977
Nuclear organizer region
McNeal et al., 1988a
Proliferative markers
BrDU
Abrahamsson et al., 1989; Ercole et al., 1987
Ki-67
Ercole et al., 1987; Shah et al., 1991; Thompson, 1990
PCNA
Sherwood et al., 1990
Ploidy S phase
Gittes, 1987; Grignon and Wright, (in press); Sporn, 1992; Stege et al., 1992
Thymidine labeling
Lovern et al., 1975
Growth factors
egfr
Eaton et al., 1988; McManus et al., 1976; Sacks et al., 1975
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Marker
References
fgf
Schiebler et al., 1992; Visakorpi et al., 1991
IGF, NGF, PDGF, KGF
Visakorpi et al., 1991
tgf
Fjellestad-Paulsen et al., 1988; Ito et al., 1988; Mellon et al., 1992; Schiebler et al., 1992; Tsukamoto et al., 1988
tnf
Fruehauf and Sinha, 1992
Oncogenes
erb (c-erbB-2)
McManus et al., 1976
myc
Schiebler et al., 1992; Voeller et al., 1991
neu (p 185)
Bostwick et al., 1994; Kuhn et al., 1993; Latil et al., 1994; Sadasivan et al., 1993; Veltri et al., 1994; Zhau et al., 1994
ras (p21)
Amico et al., 1991; Morote Robles and Ruibal Morell, 1987; Schiebler et al., 1992; Yoshiki et al., 1987
src, fos, abl
Visakorpi et al., 1991
Tumor-suppressor genes
p53
Amico et al., 1991; Epstein and Lieberman, 1985; McManus et al., 1976; Voeller et al., 1991
Rb
Amico et al., 1991
Neuroendocrine differentiation
ACTH
Bauer, 1988; Eble and Epstein, 1990; Fox et al., 1993; Fukutani et al., 1983; Hagood et al., 1991; Nemoto et al., 1990; Park et al., 1987; Purnell et al., 1984; Rubenstein et al., 1988; Sellwood et al., 1969; Tarle and Rados, 1991; Vuitch and Mendelsohn, 1981
ADH
Doctor et al., 1986; Guinan et al., 1989; Newmark et al., 1973
Aldosterone
Clar-Blanch et al., 1992
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Marker
References
alpha-HCG
Eble and Epstein, 1990
Argentaffin
Turbat-Herrera et al., 1988, Weaver et al., 1992
beta-Endorphins
Eble and Epstein, 1990; Nemoto et al., 1990
beta-HCG
Eble and Epstein, 1990; Maddy et al., 1989; Nabi et al., 1992; Sesterhenn et al., 1991; Sukumar et al., 1991; Tawfic et al., 1993; Webster et al., 1959
BOM
Helpap, 1980; Purnell et al., 1984
Bombesin (GRP)
Maddy et al., 1989
Calcitonin
Bauer, 1988; Eble and Epstein, 1990; Eskelinen et al., 1991; Fox et al., 1993; Fuse et al., 1991; Heim et al., 1977; Helpap, 1980; Maddy et al., 1989; Purnell et al., 1984; Sarkar et al., 1992
CGRP
Fuse et al., 1991
Chromogranin
Bostwick et al., 1993; Maddy et al., 1989
Corticotropin
Fox et al., 1993
Enkephalin
Eble and Epstein, 1990
Glucagon
Eble and Epstein, 1990; Maddy et al., 1989; Purnell et al., 1984
Glucocorticoids
Clar-Blanch et al., 1992
HIAA
Radjaipour et al., 1994
Lipofuscin
Weaver et al., 1992
Neuron-specific enolase
Maddy et al., 1989; Shalitin et al., 1991
Parathormone
Fuse et al., 1991; Purnell et al., 1984
Prolactin
Maddy et al., 1989
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Marker
References
Serotonin
Broder et al., 1977; Eble and Epstein, 1990; Eskelinen et al., 1991; Helpap, 1980; Maddy et al., 1989; Purnell et al., 1984; Sassine and Schulman, 1992
Somatostatin
Eaton, et al., 1988; Eble and Epstein, 1990; Eskelinen et al., 1991; Maddy et al., 1989; Purnell et al., 1984
TSH
Eble and Epstein, 1990; Maddy et al., 1989
Other proteins
amf
Coombes et al., 1974; Mahadevia et al., 1983; Watanabe et al., 1988
CA50
di Sant' Agnese and de Mesy Jensen, 1987;
Cadherins
Delaere et al., 1988; Scrivner et al., 1991
Cathepsin B
Buck et al., 1992; Guenette et al., 1994; Hasnain et al., 1992
CEA
di Sant' Agnese and de Mesy Jensen, 1987; Milani et al., 1986; Morote et al., 1990; Papapetrou et al., 1980
Cytokeratins
Boag and Young, 1992; Jarrett et al., 1964; Partin et al., 1993; Perlman and Epstein, 1990; Shulkes et al., 1991
Glutathione S-transferase
Capella et al., 1981
Inhibin
Fekete et al., 1989
leu-7
Grasso, 1952
nmp
Kleer et al., 1993
5'-Nucleotidase
Pretlow et al., 1994
Ornithine decarboxylase
Wu et al., 1994
PD-41
Bostwick, 1990
Pepsinogen II (PG II)
Grasso, 1952
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Marker
References
Polyamines
Doctor et al., 1986; Manteuffel-Cymbrorwska, 1993; Ryzlak et al., 1992
Tissue inhibitor metalloproteinases (TIMPI–III)
Baker et al., 1994; Knox et al., 1993; Stearns and Wang, 1994; Stetler-Stevenson et al., 1993
Tissue plasminogen activator (TPA)
Grasso, 1952
Type IV collagenase
Epstein and Woodruff, 1986
Blood-group antigens
A and B
Bussemakers et al.,1991
Lewis-X antigen
Bussemakers et al., 1991
Serum markers
gamma-Seminoprotein
Akimoto, et al., 1988; Anonymous, 1985; McNeal et al., 1988b; Molland, 1978; Oesterling, 1991; Tetu et al., 1989; Trapman, 1992; Wang and Kawaguchi, 1987
p2l (not ras)
Beckett et al., 1993
PAP
Bostwick, 1989; Cantrell et al., 1981; Gleason, 1990; McNeal et al., 1988b; Miki et al., 1980; Oesterling, 1991; Piana et al., 1991; Sade and Barrack, 1991; Steiner and Barrack, 1992; van Dalen et al., 1988; Weber and Rohner, 1987; Wise et al., 1965
PSA
Bookstein et al., 1993; Cantrell et al., 1981; di Sant' Agnese, 1988, 1992; Gleason, 1990; Miki et al., 1980; Nagle et al., 1991; Ohashi et al., 1987; Rojas-Corona et al., 1987; Sherwood et al., 1991; Shinoda et al., 1988; Stamey et al., 1987; Steiner and Barrack, 1992; Tinari et al., 1993; van Dalen et al., 1988; Yogi et al., 1991
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TABLE 4-6 Etiology, Evidence, and Potential Markers of Interstitial Cystitis
Evidence
Etiology
Pro
Con
Potential Markers
Infection
Finding of fastidious organisms
Failure to respond to antimicrobials; no findings of positive markers
Antibodies, genome, or immunohistochemical detection of proteins of infectious agent
Obstruction of vascular and lymphatic channels
Fibrosis, submucosal edema; onsets related to pelvic infection or operations
Experimental models do not produce IC; surgical treatments for IC will produce blockage
Fibrosis; submucosal edema
Neuropathy
Pain, focal neural inflamtion; response to amitriptyline; afferent fibers or neurotransmitters can control inflammatory response directly or through mast cells
Perineural inflammation not found in other studies; clinical therapy highly unpredictable
Focal inflammation in and around intramural and perivesical nerve bundles; neurotransmitter levels and receptors; mast cells
Endocrinopathy
Association with oophorectomy, sex bias, small numbers in premenopausal and postmenopausal women
Symptomatic response to hormonal therapy unpredictable
Endocrine receptors and levels
Psychoneurosis
Neurotic behavior pattern
Behavior seems to be a result of disease
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Evidence
Etiology
Pro
Con
Potential Markers
Inflammation
Similarities to systemic lupus and other autoimmune disorders; chronic inflammation in pathology; some responses to steroids, NSAIDS, and immunosuppressants; positive FANA at times; Ig deposits in bladder
Ig deposits nonspecific in origin; inflammation in bladder frequently not present; phenotypes of inflammatory cells in bladder; variable response to immunosuppressant therapy; relief on diversion
Immune mediators; immunecell typing; specific antibody levels
Deficiencies in bladder lining
''Leaky'' epithelium in IC, also produced in normal subjects by protamine and reversed by heparin; decreased GAG excretion in IC patients; increased GAG produced by hydro-distention; ultrastructure shows loose urothelium
GAG treatment not always effective; deficiency never demonstrated directly
Decreased GAG in urine; altered GAG on umbrella cells
Toxic substances in urine
Diversion relieves symptoms; increased serum anti-THP IgG; cytotoxic urine
Some evidence suggests defect is from inside bladder
Antibodies vs. urinary constituents
Representative terms from entire chapter:
biologic markers
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