Detection of malignancy-associated metabolites in the sera of cancer patients by electron capture gas chromatography.

A reliable test that detects malignancy and indicates response to therapy is needed. Frequency-pulsed electron-capture gas-liquid chromatography (FPEC-GLC), a selective analytical technique that is sensitive to 15 fmol quantities of metabolites, was used to analyse derivatised acidic chloroform extracts of sera from patients with biopsy-proven cancer, non-malignant infectious and non-infectious disease, and healthy controls. Two peaks designated P1 and P10, not found in serum from healthy controls (n = 7) or patients with non-malignant disease (n = 85), were detected in biopsy-proven samples (n = 52) from cancer patients. P1 and P10 were later shown by chemical and mass spectral studies to be carboxylic acids. When one or both of these peaks were detected in the sera of non-treated patients they were always associated with malignancy. In patients responding to therapy, a reduction or disappearance of these peaks was observed. Further, it was noted that P10 persisted or increased in sera of patients with progressive cancer not responding to therapy. We conclude that this test has potential in diagnosis and for following the response of the disease to therapy.

Cytokeratin shedding in urine as a biological marker for bladder cancer: monoclonal antibody-based evaluation.

Cytokeratin shedding into urine was measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) in 282 individuals. Samples included urine from normal controls, patients with urogenital conditions and bladder cancer patients. A monoclonal antibody prepared against cytokeratins extracted from a hyperkeratotic low grade squamous cell carcinoma (UNME/K1) was used in the assay. The results indicated reasonable levels of sensitivity (83%), specificity (67%) and overall accuracy (70%) in the detection of bladder cancer. The levels of sensitivity in detecting squamous and transitional cell carcinoma patients were 87 and 73% respectively. The low level of specificity was due to a high frequency of false positive results (55%) within the urogenital controls; this suggests that further immunochemical and immunohistopathological analyses of associated urothelial cytokeratins are required.

UNME/K1: an IgG2a monoclonal antibody specific to cytokeratin of human urinary bladder squamous cell carcinoma.

The main distinctive feature of carcinoma in schistosomal bladder is keratinized squamous cell carcinoma. Keratins/cytokeratins constitute a multigeneic family of structurally related polypeptide markers for the malignant state of epithelial cells. A monoclonal antibody (UNME/K1) regognizing keratins associated with squamous cell carcinoma of the human urinary bladder was generated at the Urology and Nephrology Center, Mansoura, Egypt (UNME), by fusion of spleenocytes from a BALB/c mouse immunized with a keratin extract (K1) of human squamous cell carcinoma and P3X63Ag8/U1 syngeneic myeloma cells. UNME/K1 was purified by a protein-A affinity column and was of the IgG2a type, as determined by immunoelectrophoresis and gel diffusion techniques. When tested against keratins of different types of urinary bladder tumors using enzym linked immunosorbent assay (ELISA), UNME/K1 reacted only with the high molecular weight keratin of squamous cell carcinoma and showed selectivity towards specific histopathological grades of tumors.

Studies of metabolites in diarrheal stool specimens positive for Klebsiella, Serratia, and Proteus spp. by frequency-pulsed electron-capture gas chromatography.

Diarrheal stools from infants from which Klebsiella pneumoniae, Serratia liquefaciens, and Proteus mirabilis were isolated as possible causative agents of diarrhea were studied. These stools, along with control stool specimens which were collected from infants in the same village of Tamooh (near Cairo, Egypt), were analyzed by frequency-pulsed electron-capture gas chromatography (FPEC-GC). Watery stools and formed stools, to which distilled water was added, were centrifuged, and the supernatant was extracted with organic solvents and derivatized with specific functional group reagents to form electron-absorbing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines. Results from the study showed distinct differences in FPEC-GC profiles of stools positive for K. pneumoniae, S. liquefaciens, and P. mirabilis. The major differences found were that diarrheal stools from which K. pneumoniae was isolated contained acetoin, a hydroxy acid-labeled peak F, and an unidentified amine, peak A. S. liquefaciens diarrheal stools had FPEC-GC profiles like the controls with the exception that an amine, peak A, was detected. The diarrhel stools containing P. mirabilis produced a distinct amine profile.

Cytokeratin shedding in urine: a biological marker for bladder cancer?

Cytokeratin shedding into the urine was measured using an enzyme-linked immunosorbent assay in 154 individuals. Samples include urines of normal controls, patients with urological conditions other than bladder cancer and bladder cancer patients. The assay results reflect the potential value of cytokeratin shedding in urine as a new urinary marker for bladder cancer. A 94% sensitivity for patients with squamous cell carcinoma of the bladder suggested the importance of using antibodies prepared against cytokeratin extracted from the same cell type of carcinoma as that to be detected. The relatively low sensitivity shown while detecting transitional cell carcinoma patients and the relatively low degree of assay specificity suggested the use of a panel of monoclonal antibodies specific to various types of cytokeratins of bladder carcinoma.

Schistosomal specific nephropathy leading to end-stage renal failure.

In this study 17 patients, 11 with end-stage renal failure and six with nephrotic syndrome were selected. The selection criteria were presence of active intestinal schistosomiasis and absence of any surgical or other medical disease which could explain the renal disease. When examined by light microscopy, kidney biopsies showed membranoproliferative glomerulonephritis in nine, membranous in four, focal segmental glomerulosclerosis in two, sclerosing glomerulonephritis in one case, and no changes in another case. Direct immunofluorescence showed IgG deposits in 13 cases, IgM in 10 and different complement components (C3, C1q) in eight cases. Eluates from the kidney biopsies of the 17 schistosomal as well as six control cases were examined by ELISA against schistosoma mansoni adult worm antigen (AWA). This test showed the presence of antibodies against the AWA in 12 out of 17 of the schistosomal cases, and zero out of six of the controls. When examined by direct IFA using sheep anti-circulating anodic antigen/FITC and by indirect IFA using monoclonal antischistosomal CAA IgG3, kidney biopsies of the ELISA positive cases showed granular deposits of circulating anodic antigen (CAA). We conclude that schistosomal specific nephropathy does exist in the clinical settings and can lead to end-stage renal disease, with CAA probably being a major responsible antigen.

Frequency-pulsed electron-capture gas-liquid chromatographic studies of chemical changes in sera of patients with schistosomiasis.

Sera from well documented cases of Schistosoma mansoni and S. haematobium infections as well as controls, were studied by frequency-pulsed electron-capture gas--liquid chromatography (FPEL-GLC) and mass spectrometry for detection of carboxylic acids and amines. Many carboxylic acids and unidentified peaks were detected. In a few serum specimens from infected patients, putrescine and cadaverine were detected. Indications are that in these few patients with high egg counts enough diamines were present to possibly produce amine toxicity. Following the initial investigation, the basic chloroform extractions, which contained amines, were further studied by FPEC-GLC with the aid of splitless injection and a capillary column. Several amines were detected which seemed to be related to schistosomiasis. Mass spectra were obtained on an unidentified schistosamine peak. The possible significance of the data is discussed.

Studies of metabolites in diarrheal stool specimens containing Shigella species by frequency-pulsed electron capture gas-liquid chromatography.

Eleven diarrheal stool specimens and 10 control stool specimens from Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). Four cases involving Shigella sonnei, three cases involving Shigella boydii, and four cases involving Shigella flexneri were studied. The aqueous stools were centrifuged, extracted with organic solvents, and derivatized to form specific electron-capturing derivatives of carboxylic acids, alcohols, hydroxy acids, and amines. Analyses were performed on high-resolution glass columns with an instrument equipped with an extremely sensitive electron capture detector that is specific for the detection of electron-capturing compounds. The diarrheal stools studied had specific FPEC-GLC profiles and contained metabolic markers that readily distinguished between the Shigella spp. studied and Escherichia coli producing heat-stable or heat-labile enterotoxins. S. sonnei stools contained hexanoic acid, 2-hydroxy-4-methylmethiobutyric acid, and some unidentified alcohols that distinguished this organism from other enteric pathogens. S. boydii produced an acid that was unique for this species, and S. flexneri produced alcohols that could be used to distinguish between it and other enteric organisms. The FPEC-GLC profiles obtained during this study were also very different from those reported earlier for Clostridium difficile and rotavirus. This study presents further evidence that the selectivity and sensitivity of FPEC-GLC techniques can be used to rapidly identify causative agents of diarrhea and detect physiological changes that occur in the gut during the course of diarrheal illness.

Rapid differentiation of enterotoxigenic Escherichia coli that produce heat-stable and heat-labile toxins by frequency-pulsed electron capture gas-liquid chromatography analysis of diarrheal stool specimens.

Thirty-three stool specimens from infants in the village of Tamooh near Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). In 13 of the diarrheal cases, the suspected causative agent isolated was Escherichia coli which produced heat-stable toxin (ST), and in 10 other cases E. coli that produced heat-labile toxin (LT) were isolated. Ten control stool samples, collected from infants from whom no pathogenic organisms were isolated, were analyzed at the same time. Comparisons also were made against healthy control stools from individuals in the United States who had been previously analyzed by FPEC-GLC (Brooks et al., J. Clin. Microbiol. 20:549-560, 1984). The stools were suspended in water and centrifuged, and the supernatant was extracted with organic solvents and derivatized to form electron-capturing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines. Results from the study showed distinct differences among the FPEC-GLC profiles of E. coli ST-positive stools, of E. coli LT-positive stools, and of the control stool samples. An unidentified compound appearing in the ether-soluble hydroxy acid fraction from E. coli ST-positive stools was tentatively identified by mass spectrometry as 6-methoxy-2-hydroxyhexanoic acid. 6-Methoxy-2-hydroxyhexanoic acid was found in all stools that contained E. coli ST but was not present either in stools from which E. coli LT was isolated or in control samples. 6-Methoxy-2-hydroxyhexanoic acid may prove to be an important marker for use in the identification of E. coli ST. In addition to 6-methoxy-2-hydroxyhexanoic acid, the carboxylic acid, alcohol, and amine FPEC-GLC profiles obtained from stools were very different between these two organisms. The data indicate that FPEC-GLC analysis of diarrheal stool specimens might be a rapid way to distinguish diarrhea caused by E. coli ST, E. coli LT, Clostridium difficile, and rotavirus.

Studies of stools from pseudomembranous colitis, rotaviral, and other diarrheal syndromes by frequency-pulsed electron capture gas-liquid chromatography.

Thirty-five patients with various diarrheal syndromes and 22 controls were studied. All stool samples were carefully cultured for Clostridium difficile, using selective isolation media. Cytotoxin assays with proper antitoxin neutralization were done in MRC-5 cells. The stool samples were extracted four times, three times at pH 2 and once at pH 10, using CHCl3 or ether. Derivatizations of extracts were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol, and all derivatives were analyzed by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). A dedicated computer was used to assist in both qualitative and quantitative data analysis. Isocaproic acid (iC6) was always found in stool from which C. difficile was isolated and was absent in C. difficile-negative specimens. p-Cresol was found frequently in both persons with pseudomembranous colitis and controls. Tryptamine was found in stool containing C. bifermentans. The FPEC-GLC profiles of persons with acute diarrhea were very different from those of normal persons. Diarrhea associated with adenovirus and rotavirus, Klebsiella spp., and Escherichia spp. showed different FPEC-GLC patterns. Stools from well persons consistently contained full-scale peaks of pyruvic, acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids. In rotavirus stools isobutyric, isovaleric, and valeric acids were reduced in quantity from those found in control stools, whereas propionic and butyric acids were increased.

Identification of diethylene glycol in sera from Egyptian children by frequency-pulsed electron-capture gas-liquid chromatography.

Sera taken from fifteen patients (from Kerdasa village near Cairo, Egypt) infected with Schistosoma haematobium, with eggs present in the urine, were studied by frequency-pulsed electron-capture gas-liquid chromatography (FPEC-GLC). Some of the patients were treated with metrifonate and again studied by FPEC-GLC. Diethylene glycol was detected in the sera of untreated patients infected with S. haematobium. This compound was identified by negative chemical ionization and electron-impact mass spectrometry. Initially we suspected that the build-up of diethylene glycol in these patients was caused by schistosomiasis infection. However, in a follow-up blind-coded study using FPEC-GLC, which included 37 sera from Kerdasa and Tamooh villages near Cairo, Egypt, we detected diethylene glycol in eleven samples, four of which were controls from the villages. These latter findings indicate that the source of diethylene glycol might be the environment or foodstuffs, but the specific source has not been determined. Regardless of the source, diethylene glycol could affect the health of these Egyptian children by causing a narcotic effect, increased bladder stones, and increased numbers of bladder tumours.