A freshly passed stool sample is preferred, so that the aqueous phase can be obtained as soon as possible. Facilities for collection and immediate analysis are not always available in clinical laboratories. We describe our experience in a specialized stool chemistry laboratory.

Methods

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Patients

We reviewed the records of 325 patients referred for stool chemistry analysis. Although a standard protocol was advertised, guidelines were not always followed. Thus, some patients were unable to produce specimens or submitted fully formed stools. Other samples were too small for analysis or contained barium from previous radiologic examinations. In all, samples from 212 patients were available for analysis. The complete records of these patients were reviewed, and important data were missing in 10 of them, leaving 202 patients for analysis. All had had diarrhea for at least 4 weeks. They were being investigated by different physicians, and the clinical evaluations, therefore, were not uniform. However, endoscopy of the colon with biopsy; barium radiographs; stool microscopy and culture; investigations for malabsorption; and blood chemistry and hematologic screening tests had usually been done. Particular attention was paid to the dismissal diagnoses, although any subsequent admissions and investigations were also factored into the final diagnoses (Table 1).

Factitial diarrhea was diagnosed in patients in whom laxatives were identified or in patients whose stools showed evidence of having been diluted. Functional diarrhea was diagnosed when clinical features were suggestive [6] and laboratory tests were negative. When the diarrhea was of sudden onset, a presumptive diagnosis of postinfectious diarrhea was made. Similarly, when symptom onset was closely related in time to cholecystectomy, a temporal relation was assumed.

Microscopic or collagenous colitis was diagnosed when the results of other investigations were negative and when compatible histologic changes were seen on colonic biopsy specimens [7]. A diagnosis of malabsorption was made for all patients with sprue, pancreatic insufficiency, and diarrhea after gastrectomy in whom fecal fat excretion was 7 g or more per 24 hours. Patients in the miscellaneous group had various organic diseases, including ulcerative colitis, Crohn disease, and the carcinoid syndrome.

Stool Collection and Initial Processing

More than 90% of the patients were evaluated as outpatients. Referring physicians were asked to ensure that patients were passing loose stools and that they had received no laxatives, enemas, or barium in the previous 48 hours. Patients were supplied with containers and asked to collect (and to cool or freeze) any stools passed after 5:00 a.m. the next day, and to come to the laboratory at 8:00 a.m., where additional samples would be collected. Our goal of having three individual stools for analysis was achieved for 141 patients. Each sample was weighed and stirred, and an aliquot of 50 mL was centrifuged at 3000 revolutions per minute for 15 minutes. Supernatants from each sample were analyzed in duplicate; data from the sample with the greatest wet weight from each patient are reported.

Analytic Methods

Table 2 details the analytic methods used in our study. Sodium and potassium concentrations were analyzed photometrically (Beckman Klina Flame Photometer, Beckman Instruments, Fullerton, California), and chloride concentrations were analyzed electrolytically (Corning Scientific Instruments, Medford, Massachusetts). Osmolality was measured by freezing point depression (Advanced Instruments, Inc., Needham Heights, Massachusetts), and pH was determined using a Beckman 031 pH Meter (Beckman Instruments). The osmotic gap was calculated in two ways: Twice the sum of the concentrations of sodium and potassium [mM] was subtracted 1) from the measured osmolality of stool water or 2) from 290 mOsm/kg (the osmolality of extracellular fluids, such as normal fecal water) [1].

Magnesium was quantitated using a Oxford Magnesium Stat Kit (Oxford Labware, St. Louis, Missouri) [8]. To establish positive control values, we analyzed liquid stool samples from 15 patients who had received 125 mmol of magnesium sulfate (Epsom salts). We compared the Oxford Magnesium Stat Kit and atomic absorption spectrometry; the two methods correlated well. Loose stools had a mean (±SE) magnesium concentration of 68 ±5 mM (range, 38 to 110 mM).

Phosphorus concentration was measured using a Stanbio phosphomolybdate kit (Stanbio Laboratory, San Antonio, Texas), on the basis of the method of Daly and Ertingshausen [9]. So that we could obtain positive controls, 16 healthy volunteers received a laxative dose (105 mmol) of sodium phosphate (PhosphoSoda; C.B. Fleet, Lynchburg, Virginia). Subsequently, 13 controls passed loose stools, in which the mean (±SE) phosphorus concentration was 182 ±30 mg/dL (range, 31 to 417 mg/dL). Sulfate was determined semiquantitatively by adding barium chloride to acidified supernatant and observing the barium sulfate precipitate.

The presence of phenolphthalein was determined qualitatively by adding 0.2N NaOH to the fecal aliquot. If a pink color was seen, the presence of phenolphthalein was confirmed by extracting the sample with ethyl ether, redissolving it in aqueous NaOH, and identifying phenolphthalein by spectrophotometric absorption at 540 nm [10]. Anthraquinone laxatives (such as senna and cascara) also produce a pink color with alkalinization. Using positive controls for tests for senna and cascara, a urine sample was extracted using carbon tetrachloride and was alkalinized at pH 12 with ammonium hydroxide. Pink color in the ammonia phase was taken to indicate the presence of the drug [11]. Sensitivities were tested by additional dilution of positive controls; these standards had been based on a therapeutic amount of each drug. Further dilution of phenolphthalein (20-fold) and senna and cascara (5-fold) still yielded positive results. When laxative abuse was strongly suspected, stool supernatants and urine samples were analyzed with mass spectroscopy for the presence of bisacodyl [12].

Results

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Factitial Diarrhea

Evidence of laxative use or of stool dilution was present in 35 patients (17% of analyzable patients). Those using laxatives fell into three categories: those with stools, urine, or both that contained phenolphthalein (n = 9) or bisacodyl (n = 2); those with stool supernatants that had magnesium concentrations of 50 mM or more (n = 7); and those with stool supernatants with magnesium concentrations of 25 to 49 mM (n = 12) but with supporting evidence of magnesium-induced diarrhea, such as positive fecal sulfate levels or history of a strong relation between magnesium ingestion and diarrhea. Five patients submitted hypotonic stools (Table 3); they were assumed to have mixed their stools with water or another hypotonic fluid.

Stool compositions of patients ingesting secretory laxatives were compared with those of patients with osmotic diarrhea attributed to magnesium (Table 4). The mean fecal (Na) was significantly less in patients with osmotic diarrhea (P < 0.01) than in those taking secretory laxatives, and the osmotic gap was greater (P < 0.01).

Functional Diarrhea

In all three subgroups of patients with functional diarrhea, the usual pattern was for (Na) to be high and for (K) to be low; the mean osmotic gap was 35 ±5 mOsm/kg, implying that these patients had a predominantly secretory diarrhea.

Collagenous and Microscopic Colitis

High mean concentrations of sodium (84 ±5 mM), low mean concentrations of potassium (44 ±4 mM), and small osmotic gaps (39 ±5 mOsm/kg) were consistent with a secretory process; moreover, collagenous and microscopic colitis were not different (P > 0.05). A few patients with Crohn disease or ulcerative colitis (n = 9) had results similar to those of patients with microscopic or collagenous colitis.

Malabsorption and Miscellaneous Diagnoses

No consistent, significant changes were noted. However, steatorrheal stools clustered into two groups: those with large (>100 mOsm/kg) and those with small (<50 mOsm/kg) osmotic gaps (Figure 1). Few samples from any group had pH values of less than 6.0; the lowest pH readings were in the patients with steatorrhea (Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 1. The osmotic gap of stool water for subgroups of patients with diarrhea. The osmotic gap is derived from the following formula: 290 – (2[sodium concentration + potassium concentration]). Patients with negative values were not included, nor were the patients with miscellaneous diagnoses. All patients with fecal fat excretion of more than 7 g/d were included, regardless of whether an underlying diagnosis was established (see Table 1). Values for osmotic gaps between 50 and 100 mOsm/kg (dotted lines) represent a "gray zone" between low (secretory diarrhea) and high (osmotic diarrhea) gaps. Micro/Collag = microscopic and collagenous.

View larger version (17K): [in this window] [in a new window]   Figure 2. The pH of stool water for subgroups of patients with diarrhea. The group with miscellaneous diagnoses was not included. All patients with fecal fat excretion of more than 7 g/d were included, regardless of whether an underlying diagnosis was established (see Table 1). Values for pH between 5.0 and 6.0 (dotted lines) are similar to those reported for experimental carbohydrate malabsorption diarrhea [15]. Micro/Collag = microscopic and collagenous.

Discussion

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Diarrhea of brief duration is extremely common, but chronic symptoms (lasting 4 or more weeks) require evaluation. Indeed, obscure ("functional") diarrhea is a major reason for referral to gastroenterologists [13]. Because of the numerous causes of chronic diarrhea [1], patients with this symptom often require expensive and invasive investigations. Attempts have therefore been made [1, 14] to classify the pathophysiologies of diarrhea.

Chronic diarrheas have been subdivided into 1) those with osmotic causes, in which small molecules [such as unabsorbed dietary substances and poorly absorbed ions] retain water in the bowel lumen; 2) secretory states, in which the intestinal mucosa is stimulated [for example, by bacterial toxins, bile acids, or fatty acids] to secrete ions and water; 3) disorders of transit, which reduce the exposure of chyme to the absorptive surface; and 4) exudative states [1, 14]. The usefulness of these classifications, which have led to proposals whereby the composition of stool water could help to identify these mechanisms [1], was confirmed when experimental diarrhea was induced in healthy persons [15-19].

However, clinical diarrhea involves several pathophysiologic mechanisms [1]; furthermore, the collection of samples is usually less well controlled in the clinic than in the experimental laboratory. Our experience in comparing three separate stool samples from the same day (data not shown) confirmed that individual stools sometimes varied widely in composition. Part of this variability is caused by bacterial fermentation of carbohydrate, which continues even after stools are passed; this increases osmolality and decreases pH level. Fermentation is slowed, but not stopped, by refrigeration [15]. Despite these complications, our findings confirm the usefulness of the general classification of pathophysiologies of diarrhea [1, 14].

When diarrhea was caused by secretory laxatives (such as phenolphthalein or bisacodyl), the osmotic gap was small; in patients with elevated magnesium levels, the gap was much greater (Table 4). Patients with collagenous or microscopic colitis had a predominantly secretory diarrhea with low osmotic gaps. Similarly, patients with functional diarrheas had features compatible with secretion. In one study of a group of 40 patients with the irritable bowel syndrome [20], 83% had evidence of bile acid malabsorption. Our patients with generalized malabsorption showed a broad range of osmotic gaps with a bimodal distribution (Figure 1). We speculate that some had secretory states, possibly due to unabsorbed fat, whereas others had osmotic diarrhea caused by carbohydrate malabsorption. We could not distinguish a group with isolated carbohydrate malabsorption large enough to define this pathophysiology adequately.

The pH level of fecal water showed only very broad trends; overlap among groups was large but, as predicted, patients with malabsorption had the lowest pH levels. Fecal short-chain fatty acids result from the anaerobic metabolism of carbohydrate residues. However, the results of our pH tests were not as clear-cut as the results produced when diarrhea was induced experimentally using lactulose [15], but they were more in accord with previous findings in patients [1].

We used simple techniques to test for common, over-the-counter laxatives, including phenolphthalein, senna and other anthraquinones, magnesium, sulfate, and phosphate. If laxative use is strongly suspected, we also recommend specific drug analysis for bisacodyl using mass spectroscopy and gas chromatography [12]. Fine and colleagues [17] established a cutoff of 50 mM for diarrheogenic concentrations of magnesium in stool water. For pure magnesium-induced diarrhea, magnesium concentrations were greater than 100 mM [17], but they were lower when magnesium and phenolphthalein were taken together. Fine and colleagues concluded that water secretion, induced by phenolphthalein, diluted the concentration of magnesium in stool water. After a single laxative dose of magnesium sulfate, we found a mean magnesium concentration of 68 mM (range, 38 to 110 mM). Thus, we felt secure in diagnosing magnesium-induced diarrhea when samples contained more than 50 mM of magnesium; we were more reticent when samples contained 25 to 50 mM magnesium. Our experience with phosphate and sulfate is more limited, but the presence of sulfate together with elevated levels of magnesium suggests the ingestion of magnesium sulfate (Epsom Salts). Moreover, the presence of elevated fecal levels of sulfate or phosphate might explain an otherwise obscure diarrhea when a major osmotic gap is absent [18].

The group of patients with hypotonic stools was small but well defined. The colon does not excrete free water [21]; thus, fecal hypotonicity indicates the addition of water, urine, or another hypotonic fluid. Under extremes of diuresis, urine can achieve an osmolality of 150 mOsm/kg [22], but osmolality does not usually decrease to less than 250 mOsm/kg, especially if diarrhea has resulted in volume depletion. Thus, any appreciable decrease in fecal osmolality suggests the addition of fluid to fecal samples, as might be done by patients who have the Munchausen syndrome or are malingering [21]. In four of our five patients, one of these diagnoses was suggested by the records.

Fecal water analysis has a definite but limited role in elucidating the pathogenesis of diarrhea. Even though we established guidelines for fecal collections, one third of all referrals yielded samples unsuitable or inappropriate for analysis. Moreover, the utility of this approach to the management of individual patients is limited. However, of this selected group of 202 patients, 35 (17%) had evidence of factitial diarrhea, supporting the argument that stool analysis is a useful and inexpensive screening test for obscure diarrheas [2-5]. The cost of these tests (<$350) is far less than that of repeated invasive, potentially dangerous investigations. The methods are not demanding and could be established in any hospital center.