Anti-tissue transglutaminase antibodies and their role in the investigation of coeliac disease.

Coeliac disease (CD), caused by an inappropriate T-cell-mediated immune response to the ingestion of cereal proteins in genetically susceptible individuals, is a common disorder with a prevalence of about 1% in Caucasian populations. It has a strong association with other autoimmune disorders, particularly type 1 diabetes and autoimmune thyroid disease. Although primarily affecting the small bowel, CD is a multisystem disorder and the adult or child patient may initially present to a wide range of clinical specialties. The concept of the 'coeliac iceberg' has been used to emphasize that many cases currently remain undiagnosed. The identification of tissue transglutaminase (TGA)-2 as the antigen against which the autoantibodies are directed has led to a greater understanding of the pathogenesis of CD and to the development of improved serological tests. Enzyme-linked immunoassays using human tissue TGA as antigen have high diagnostic sensitivity and specificity for the detection of CD. This review examines the evidence for adopting IgA anti-tissue TGA as the first-line diagnostic test for CD. It recommends a laboratory algorithm for the use and interpretation of TGA to enable the clinical laboratory to play a full part in detecting and monitoring a disorder that is eminently treatable once the diagnosis has been considered and confirmed.

Partial purification and some properties of gamma-glutamyl transpeptidase from human bile.

1. Gamma-Glutamyl transpepetidase ((5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2) from human bile has been partially purified using protamine sulphate treatment, DEAE-cellulose chromatography and Sephadex G-200 filtration. The procedure resulted in 150-fold increase in specific acitivity with a 37% yield. 2. The partially purified enzyme showed a single zone of enzyme activity by polyacrylamide gel electrophoresis and eluted in the inner volume of Sephadex G-200. 3. The enzyme had a pH optimum of 8.1 and Km of 1.52 mM using gamma-glutamyl p-nitroanilide as substrate. 4. The effects of cations and different gamma-glutamyl acceptors on the activity of the enzyme are reported. 5. As bile gamma-glutamyl transpeptidase appears to be soluble in the absence of detergents, it is suggested that bile may prove to be a useful source for further studies of the kinetic properties and physiological role of human gamma-glutamyl transpeptidase.

Plasma lipids in tropical sprue.

Previous studies indicated that many patients with tropical sprue in southern India have triglyceride accumulation within the cells of the intestinal mucosa. This could be due to essential fatty acid deficiency as a result of steatorrhea in subjects on a diet normally low in linoleic acid. Plasma lipids have, therefore, been studied in patients with tropical sprue and the results compared to values observed in healthy controls. The low intake of linoleic acid was found to be adequate to maintain normal levels of arachidonic acid and to repress the formation of 5,8,11-eicosatrienoic acid in rural controls. Patients with tropical sprue had decreased levels of plasma linoleic acid, raised monoenoic fatty acids and the appearance of 5,8,11-eicosatrienoic acid in the lecithin fraction. All of these observations indicate essential fatty acid depletion although unequivocal evidence of essential fatty acid deficiency was not present in any of the patients. The degree of essential fatty acid depletion observed is unlikely to be the cause of the mucosal accumulation of triglyceride in tropical sprue.

An interpretation of the elevation of serum alkaline phosphatase in disease.

Evidence is presented in this paper which supports the hepatogenic theory for the mechanism by which the level of serum alkaline phosphatase is raised in liver disease and provides additional evidence that serum phosphatase is not excreted in the bile. By starch gel and paper electrophoresis the normal serum alkaline phosphatase isoenzyme is shown to be rarely present in hepatic bile. The action of neuraminidase demonstrates that beta-globulin isoenzymes of liver and bone are not identical. From these results a theory which clarifies the rationale of the elevation of alkaline phosphatase in bone and liver disease is postulated. The proposed mechanism may be summarized as follows. The normal serum level is the result of two factors, the rate of release of the enzyme from the tissues, principally liver and bone, and the rate of inactivation of the enzymes in the serum and body protein pool. In osteoblastic bone disease the elevated level is due to the rate of release of the enzyme exceeding the rate of inactivation. The raised level does not indicate an inability of the liver to excrete the enzyme via the biliary tract. In liver disease the increase in serum levels is a result of increased liberation of the enzyme from the sinusoidal surface of the liver cell and of regurgitation of the biliary isoenzyme back into the serum.

The nature of the alkaline phosphatases of bile.

The main alkaline phosphatase isoenzymes of human bile have been purified by DEAE-cellulose chromatography. Characteristics of the isoenzymes, such as electrophoretic mobility before and after butanol extraction, Michaelis constant, and change in electrophoretic mobility following exposure to neuraminidase have been studied and compared with isoenzymes from other sources. The results show that the main alkaline phosphatase of bile is derived from the liver. It is present as a protein phosphatidylcholine complex.

One-hour serum xylose as an absorption test in the tropics.

The 1-hour serum xylose (surface area corrected) as an indication of xylose absorption after 5 g oral D-xylose has been compared with the 5-hour urine excretion test in a tropical population. The study confirmed that the peak serum xylose concentration occurs at 1 hour and that correction to a constant body surface are improves the discrimination between subjects with normal and impaired xylose absorption. The significantly lower reference range for the 1-hour surface area corrected serum xylose (0.55-1.11 mmol/l) compared to the UK figure reflects the reduced absorptive capacity of the jejunum, a result of tropical enteropathy. In view of the difficulties in obtaining accurate urine collections in tropical countries, especially in field studies, it is recommended that the 1-hour serum xylose (surface area corrected) should be adopted as the standard test of xylose absorption.

The prevalence of coeliac disease in adult diabetes mellitus.

Coeliac disease occurs more commonly in children with insulin-dependent diabetes mellitus (IDDM) than in the general population, but the prevalence of coeliac disease in adults with diabetes is unknown. We therefore screened an adult hospital-based diabetic population using IgA antigliadin antibody (IgA-AGA) to identify those patients requiring intestinal biopsy. In 1 year, 1789 patients (43% IDDM, 57% NIDDM) were screened, and 73 had raised IgA-AGA. Of these patients, 49 agreed to duodenal biopsy and 13 (10 IDDM) had coeliac disease. Selective IgA deficiency was found in eight patients, one of whom had coeliac disease. Of these 14 patients with newly diagnosed coeliac disease, four had microcytic anaemia, nine a low serum ferritin, and four a low albumin-corrected calcium. Eight patients had symptoms which improved on gluten withdrawal. Dietary compliance was maintained in 6/8 symptomatic patients, but only in 1/6 without symptoms. Included in the 1789 patients were four (all IDDM) with known coeliac disease. The overall prevalence of coeliac disease in adult patients with IDDM was 1:50 compared with 1:340 in NIDDM. Coeliac disease is common in adults with IDDM and may cause malabsorption and ill health. It should be suspected in any IDDM patient with gastrointestinal symptoms or unexplained anaemia.

Determination of sulphur compounds in beer using headspace solid-phase microextraction and gas chromatographic analysis with pulsed flame photometric detection.

A simple and sensitive method for the analysis of volatile and semi-volatile sulphur compounds in beer at trace levels was developed using headspace solid-phase microextraction (SPME) and gas chromatography with pulsed flame photometric detection. Different SPME fibres were tested and a Carboxen-polydimethylsiloxane coated fibre was found to be the most appropriate. The adsorption and desorption conditions were optimised. The effect of ethanol concentration in the sample on the extraction of analytes was examined. A 60 m non-polar capillary column preceded by a 10 m length of a polar column was found to be capable of separating a wide range of C1-C6 sulphur compounds. The pulsed flame photometric detector enabled increased sensitivity to be obtained over previous methods, such as dynamic headspace followed by conventional flame photometric detection or sulphur chemiluminescent detection, with high sulphur selectivity. Two sulphur compounds, 2-methyl-1-butanethiol and 3-methylthiophene, were identified in beer for the first time.

A UK survey of laboratory-based gastrointestinal investigations.

A survey of hospital laboratory services has demonstrated marked deficiencies in the performance of gastrointestinal function tests. The repertoire of gastrointestinal investigations available varies widely between laboratories and, in general, analyses are performed infrequently. Most laboratories do not perform internal quality control, and inter-laboratory reproducibility of some analytes is very poor. A wide variety of protocols and reference ranges are in use, many of which are unevaluated. Some analytical methods and protocols in current use are outdated, with published improvements not being applied.

Bromocresol purple and the measurement of albumin. Falsely high plasma albumin concentrations eliminated by increased reagent ionic strength.

This study was undertaken to investigate the extent of the reported discrepancy between serum and plasma albumin concentrations measured by the bromocresol purple (BCP) method. We have shown that plasma albumin concentrations are falsely increased by turbidity due to the precipitation of fibrinogen when plasma is diluted into the BCP reagent. Modification of the BCP buffer (to 0.1M acetate containing 0.15M NaCl, pH 5.5) eliminates the turbidity and thus permits the measurement of albumin in either serum or plasma. Plasma albumin concentrations measured with the modified BCP reagent show excellent correlation with results by an immunoturbidimetric method.

A comparison of automated fructosamine and HbA1c methods for monitoring diabetes in pregnancy.

Two automated methods for measuring fructosamine (Test Plus and the original fructosamine assay) and glycated haemoglobin (Tina-quant immunoassay) were compared to determine which is the best index of blood glucose control during pregnancy. Thirteen women with type 1 diabetes were studied, with four-weekly measurements of HbA1c and fructosamine Test Plus using a Hitachi 911 analyser and fructosamine measured using an Olympus AU800 analyser. HbA1c correlated better (r = 0.573) with mean blood glucose (MBG) concentration than did fructosamine Test Plus (r = 0.347), even after correction for total protein concentration (r = 0.463), while there was no significant correlation with the original fructosamine method (r = 0.201). HbA1c correlated better with fasting/pre-prandial MBG concentrations, whereas fructosamine Test Plus correlated better with post-prandial MBG concentrations. Fructosamine Test Plus decreased with gestational age, and correlated with albumin and total protein concentrations, whereas HbA1c did not change with gestational age. Thus, HbA1c and fructosamine Test Plus were found to be useful in verifying home blood glucose measurements in diabetic pregnancy, with HbA1c being the best predictor of MBG concentration.

Rapid enzymatic method for the measurement of mannitol in urine.

A previously described method for mannitol in urine has been modified and improved. End product inhibition by fructose in the mannitol dehydrogenase method for mannitol has been minimized; the assay is linear over a sample mannitol concentration range of 0-12 mmol/L; no significant interference from other sugars or sugar alcohols could be demonstrated. The method is precise (within-batch CV less than 1%), rapid and shows excellent recovery of mannitol in spiked samples. Comparison with gas liquid chromatography shows excellent correlation (r = 0.994) between the two methods.

IgA-antitissue transglutaminase: validation of a commercial assay for diagnosing coeliac disease.

We have evaluated a commercial assay for serum IgA class antibodies to tissue transglutaminase, the enzyme identified as the major endomysial autoantigen in coeliac disease (CD). Sera were available from 130 adults diagnosed with CD in Southern Derbyshire between 01 01 97 and 31 12 99. Sera from 100 patients without villous atrophy on small intestinal biopsy were controls. The ability of the assay to detect abnormally low total IgA levels was assessed using sera from 18 subjects with IgA deficiency. Sensitivity and specificity of this IgA-anti tissue transglutaminase (tTGA) assay (86.2%, 91.0%) were inferior to endomysial antibody (EMA; 93.8%, 100%). tTGA has significantly higher sensitivity than IgA-antigliadin (76.2%). tTGA was appropriately undetectable (<0.03 U/mL) in 17 of 18 subjects with selective IgA deficiency. The high likelihood ratio (35) for tTGA at levels >9.0 U/mL and methodological advantages over EMA suggest that tTGA could be used as a first line diagnostic test for CD. At tTGA levels of 4-9 U/mL, use of EMA as a second line test would improve specificity.

Correlated clinical, histopathological, HBV and AFP studies of primary carcinoma liver.

This paper presents the clinical and histopathological features of 221 Primary Carcinoma Liver studied over the last eight years. HBsAg studies were carried out in 93, AFP studies in 87 and both HBsAg and AFP in the same serum samples in 78. HBsAg positivity rate by CIEP was 29% and AFP positivity was 52%. There appears to be greater AFP posivity in HBsAg positive group compared to HBsAg negative group. Correlation of cell type with AFP positivity reveals significant greater positivity in clear type. The need for indepth studies to use morphological appearances of cancer cells as simple marker of altered function is suggested. Anti e was positive in 40% of HBsAg positive cases studied for e system, while e antigen was positive in 32%. A very unusual finding of e antigen and Anti e present at the same time in a patient with primary liver carcinoma is documented.