Metabolism of food-derived heterocyclic amines in human and rabbit tissues by P4503A proteins in the presence of flavonoids.

The ability of human and rabbit gastrointestinal-tract microsomes to metabolize the heterocyclic amine 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to a mutagen was determined with the Ames test. When human jejunal and ileal microsomes were used as the metabolic activation source, MeIQ produced 1675 and 388 revertants/mg of microsomal protein, respectively, and this increased to 29,230 and 17,963 revertants/mg of microsomal protein, respectively, in the presence of 100 microM alpha-naphthoflavone. MeIQ in the presence of control rabbit duodenal, jejunal, and ileal microsomes produced 2304 +/- 1018, 988 +/- 386, and 444 +/- 134 (mean +/- SD, four samples) revertants/mg of microsomal protein, respectively. In the presence of alpha-naphthoflavone (100 microM), these activities increased greater than 7-fold. P4503A proteins were detectable on Western blots of microsomes prepared from both human and rabbit small intestine. Further, rifampicin-induced rabbit hepatic-microsomal activation of MeIQ was completely inhibited at low concentrations of alpha-naphthoflavone, but at higher concentrations (i.e., 100 microM) this returned to control levels. Flavone also caused a marked stimulation of MeIQ activation in human and rabbit gastrointestinal-tract microsomes. The aforementioned data suggest that flavonoids markedly increase the ability of P4503A isozymes to activate heterocyclic amines to mutagens in the Ames test.

Glycosylated hemoglobins and glycosylated plasma proteins in the diagnosis of diabetes mellitus and impaired glucose tolerance.

Total and stable glycosylated hemoglobins and glycosylated plasma proteins were determined on 53 patients referred for a glucose tolerance test. Significant correlations were found with fasting blood glucose (r greater than 0.89), 2-h glucose (r greater than 0.69), and area under the glucose tolerance curve (r greater than 0.75), but the correlations with labile glycosylated proteins were not significant. Thirty-one of the patients were normal, five had impaired glucose tolerance (IGT), and seventeen diabetes mellitus (DM) according to the WHO criteria. Comparison of the glycosylated protein values showed that, in all cases, the values for those with IGT and DM were significantly (P less than 0.001) greater than the values for normals. The range of values of stable glycosylated hemoglobins for those with DM (9.4-24.4%), those with IGT (8.6-10.0%), and normals (5.0-8.5%) shows that there was no overlap between overt diabetic subjects and normal subjects. This was also found for total glycosylated hemoglobins. The results for glycosylated plasma proteins, total and stable, were comparable, but one patient with overt DM and two with IGT had values within the normal range. The measurement of glycosylated hemoglobins and glycosylated plasma proteins by the simple, precise, affinity-chromatography method is potentially a quick, accurate, and simple screening test for patients with DM and IGT and deserves consideration as criteria for their diagnosis.

Light chain nephropathy.

In 13 specimens of renal tissue from 11 patients, deposits of monoclonal immunoglobulin light chains and continuous granular electron-dense material within tubular basement membranes and in association with the glomerular basement membrane were identified. All but one patient were men n the fifth to seventh decades of life, and each presented with azotemia and features of glomerular rather than tubulointerstitial disease. Osteolytic bone lesions occurred in only three patients, and a bone marrow plasmacytosis greater than 30 percent consistent with plasma cell myeloma was identified in only four patients. Light chain distribution in the nephron was confirmed with immunoelectron microscopy and was not associated with deposition of other serum proteins such as immunoglobulin heavy chains, complement, transferrin, alpha 2 macroglobulin and albumin. The electron dense deposits differed in distribution and character from those associated with membranoproliferative glomerulonephritis type II (dense deposit disease), amyloidosis, cryoglobulinemia, macroglobulinemia and benign monoclonal gammopathy. Serum from six of these patients did not bind to normal human or rat renal parenchyma in vitro. Kappa light chain nephropathy was characterized by predominant linear tubular basement membrane kappa deposits, and nodular mesangial and linear glomerular basement membrane kappa immunostaining. Lambda light chain nephropathy was characterized by linear lambda glomerular basement membrane and tubular basement membrane immunostaining. Manifestations of glomerular dysfunction dominated the clinical presentation of light chain nephropathy, and most patients did not have typical features of multiple myeloma. The diagnosis was predicated upon thorough immmunohistologic assessment of renal biopsy material.

The effects of dietary iron overload on fumonisin B1-induced cancer promotion in the rat liver.

The present study was performed to determine whether excess hepatic iron modulates the cancer-initiating and promoting properties of FB1. Thirty-eight male F344 rats were divided into four dietary treatment groups: (i) control diet (AIN, n = 8); (ii) FB1 250 mg/kg diet (FB1, n = 10); (iii) 1-2% carbonyl iron (CI, n = 10); or (iv) FB1 plus iron loading (FB1/CI, n = 10) for 5 weeks (2 x 2 factorial design). Hepatic iron concentrations in iron-loaded animals at 5 weeks were 444 +/- 56 (CI) and 479 +/- 80 micromol/g dry weight (FB1/CI) (mean +/- SEM). All the FB1-fed rats, in the presence or absence of CI, developed a toxic hepatitis with a 4-fold rise in serum alanine transaminase (ALT) levels. FB1 appeared to augment iron-induced hepatic lipid peroxidation, as measured by the generation of thiobarbituric acid reacting substances (TBARS) in liver homogenates (P < 0.0001). Morphometric analysis showed that FB1 caused a significantly greater mean +/- SEM number of 'enzyme-altered' foci and nodules per cm2 (5.34 +/- 1.42 vs. 1.50 +/- 0.52, P < 0.05), as well as a greater area (%) of liver occupied by foci and nodules (0.33 +/- 0.12% vs. 0.05 +/- 0.03%, P < 0.001), compared with FB1/CI. The addition of FB1 to dietary iron loading caused a shift in distribution of iron from hepatocytes to Kupffer cells, probably due to phagocytosis of necrotic iron-loaded hepatocytes. In conclusion, (i) FB1 appears to cause toxicity in the liver independently from effects on lipid peroxidation; (ii) FB1 has a potentiating effect on iron-induced lipid peroxidation; and (iii) dietary iron loading appears to protect against the cancer promoting properties of FB1, possibly due to a stimulatory effect of iron on hepatocyte regeneration.

Chronic glomerular microangiopathy and metastatic carcinoma.

Two patients with metastatic colonic adenocarcinoma developed deterioration of renal function six and nine months after the diagnosis of malignant disease. A renal biopsy specimen in one case and both postmortem specimens revealed thickening of glomerular capillary loops with focal reduplication of basement membrane-like material. Ultrastructural examination of all three specimens demonstrated a lucent subendothelial zone and no evidence of electron dense deposits. Antifibrinogen staining outlined most capillary loops in one case. It appears that chronic intravascular coagulation induced by the neoplasm was the major pathogenetic process involved in the production of the glomerular lesion in each case.

Revascularization of the kidney after occlusion of the aorta and both renal arteries.

A patient who developed acute renal artery thrombosis as a complication of distal abdominal aortic occlusion is described. Because of the presence of an extensive collateral arterial supply, the right kidney survived and revascularization was accomplished successfully with a saphenous vein graft interposed between the superior mesenteric and the right renal arteries. Criteria for revascularization of renal artery occlusion are presented, with emphasis on the importance of collateral circulation and the elective correction of distal aortic thrombosis.

Creatinine filtration, secretion and excretion during progressive renal disease. Modification of Diet in Renal Disease (MDRD) Study Group.

The Modification of Diet in Renal Disease (MDRD) Study is a multicenter, randomized, controlled trial to determine acceptance, safety, and efficacy of low protein and phosphorus diets in patients with progressive renal disease. During the feasibility phase, 96 patients aged 18 to 75 years, with previously declining reciprocal serum creatinine concentration (1/PCr) and current glomerular filtration rate (GFR) from 7.5 to 80 ml/min/1.73 m2, were randomly assigned four study diets. After randomization, 91 patients were followed for a mean duration of 14.1 months. GFR, 1/PCr and creatinine clearance (CCr) were measured every three months. In an earlier report, we demonstrated relatively weak correlations of rates of change in GFR and 1/PCr during the feasibility phase; the proportion of variability in 1/PCr slopes that was explained by variability in GFR slopes (r2) was only 0.49 to 0.55. In this study, we examined the relationship of GFR and 1/PCr to other determinants of the serum creatinine concentration, including filtration (GFCr), secretion (TSCr), and total renal excretion (UCrV) of creatinine. Our results show that these parameters varied widely among individuals and changed over time. These findings may explain, in part, the relatively weak correlations. These results strengthen our previous suggestion that the rate of change in 1/PCr may not be an accurate index of the rate of change in GFR and raise questions about the validity of conclusions from other studies in which the efficacy of dietary modification in retarding the progression of renal disease was based principally on measurements of 1/PCr.

Localization of N-acetyltransferases NAT1 and NAT2 in human tissues.

Human acetyl coenzyme A-dependent N-acetyltransferase (EC 2.3.1.5) (NAT) catalyzes the biotransformation of a number of arylamine and hydrazine compounds. NAT isozymes are encoded at 2 loci; one encodes NAT1, formerly known as the monomorphic form of the enzyme, while the other encodes the polymorphic NAT2, which is responsible for individual differences in the ability to acetylate certain compounds. Human epidemiological studies have suggested an association between the "acetylator phenotype" and particular cancers such as those of the bladder and colon. In the present study, NAT1- and NAT2-specific riboprobes were used in hybridization histochemistry studies to localize NAT1 and NAT2 mRNA sequences in formalin-fixed, paraffin-embedded human tissue sections. Expression of both NAT1 and NAT2 mRNA was observed in liver, gastrointestinal tract tissues (esophagus, stomach, small intestine, and colon), ureter, bladder, and lung. In extrahepatic tissues, NAT1 and NAT2 mRNA expression was localized to intestinal epithelial cells, urothelial cells, and the epithelial cells of the respiratory bronchioles. The observed heterogeneity of NAT1 and NAT2 mRNA expression between human tissue types may be of significance in assessing their contribution to known organ-specific toxicities of various arylamine drugs and carcinogens.

Can citrate therapy prevent nephrolithiasis?

This prospective study was done to help determine whether or not oral citrate therapy can elevate urinary citrate levels and reduce the incidence of recurrent calcium stones in patients with hypocitraturia (urinary citrate less than 320 mg/24 hr). The treatment group (Group I) consisted of 10 patients treated with oral citrate and hydration. The control group (Group II) was treated with hydration alone. In Group I, the new stone formation rate (stones per patient year) fell from 1.17 +/- 0.26 to 0.45 +/- 0.32 (p = 0.07) and the twenty-four-hour urinary citrate excretion rose from 69 +/- 14 mg to 473 +/- 96 mg (p = 0.002). In Group II, the new stone formation rate fell from 0.9 +/- 0.25 to 0.27 +/- 0.13 (p = 0.03). The twenty-four-hour urinary citrate excretion increased, though not significantly, from 166 +/- 21 mg to 326 +/- 77 mg (p = 0.06). We conclude that oral citrate therapy can significantly increase urinary citrate levels in patients with recurrent stones associated with hypocitraturia but that oral citrate therapy and hydration were no better than hydration alone in reducing the incidence of recurrent stones.

Measurement of glycosylated haemoglobins using an affinity chromatography method.

After removal of the labile material, we have measured the stable glycosylated fraction of haemoglobin with a new, commercially available, phenylboronic acid affinity gel, Glycogel B. The mean value was established for 61 non-diabetics as 7.31 (SD +/- 0.92)% and for 108 diabetics as 12.70 (SD +/- 2.88)%. The method is highly reproducible with a coefficient of variation below 2.0%. The effect of changing the temperature from 7 degrees C to 37 degrees C, and pH from 8.1 to 8.9 was investigated. For accurate results the temperature should be maintained between 20 degrees C +/- 1 degree C, and the pH between 8.6 +/- 0.1. A poor, but significant correlation (r = 0.43) between glycosylated haemoglobin and simultaneous blood glucose was shown. There was a good correlation with the agar gel electrophoretic method (r = 0.95). The slope of the regression line was 1.20 which indicates that this affinity method measures more than just HbA1. The affinity method appears to offer selectivity for diabetics than the electrophoretic method.

Hepatic stem cells: a review.

The existence of a liver stem cell population has only gained credence recently, following the results of animal experiments. These cells are thought to reside in the terminal bile ductules (canals of Hering). Hepatocyte division is responsible for liver regeneration after most causes of injury. However, stem cells may contribute to hepatocyte regeneration, or even take over this role if the liver injury is severe and associated with an impairment of hepatocyte proliferation as in cirrhosis or submassive/massive necrosis, due to drugs, toxins or viruses. "Oval" cells are the descendants of the stem cells and are found in the portal and periportal regions in experimental animals within days of the liver injury. These cells proliferate to form narrow ductules, which may stain positively for biliary cytokeratins CK 19, and radiate out into the damaged parenchyma. Both in vitro and in vivo animal studies now suggest that oval cells can differentiate into bile ductular cells or hepatocytes to allow repopulation of the injured liver. As the oval cells differentiate into hepatocytes they may show positive staining for pyruvate kinase isoenzyme L-PK, albumin and alpha-fetoprotein. There is also growing evidence that bone marrow stem cells may contribute to liver regeneration. The possible involvement of hepatic stem cells in the development of dysplastic nodules, hepatocellular carcinoma and cholangiocarcinoma has been suggested but remains highly controversial. Oval cell isolation and culture techniques, together with stem cell transplantation strategies, may in the future provide novel treatments for individuals with inherited and acquired hepatic disorders.

A technique for obtaining repeated liver biopsies from rats.

Many morphological, pharmacological and toxicological studies of hepatotoxicity require frequent sampling of liver over time. In the past this has been achieved by including large numbers of animals in the study and killing subgroups at different times. In this paper we describe a technique for repeated liver biopsy that procures sufficient liver tissue for histopathological assessment and for additional studies, for example measurement of hepatic iron concentration or vitamin A measurement. The advantages and disadvantages of this technique are discussed.

Use of artificial sweeteners to promote alcohol consumption by rats.

Cirrhosis may be reliably produced in rats by exposing them intermittently to low levels of carbon tetrachloride vapour while feeding alcohol in the Lieber-DeCarli liquid diet. Providing the alcohol in drinking water that has been sweetened with sucrose is a cheaper and more convenient method but it does not yield reliable results. This study aimed to determine whether alcohol in drinking water sweetened with artificial sweeteners would give adequate alcohol intake to achieve the desired hepatic effects. Rats were fed alcohol (8% v/v) in drinking water sweetened with sucrose (5% w/v) (n = 12), or with one of the artificial sweeteners aspartame (0.025%), saccharin (0.025%) or cyclamate (0.05%) (n = 8 per agent). During the alcohol treatment the animals were exposed to carbon tetrachloride vapour, 40 ppm, six hours per night for five nights per week, over a period of 14 weeks. All groups achieved good alcohol intakes of 5-6 g/kg/day. Only one rat, in the aspartame group, became cirrhotic; all the others had varying degrees of fibrosis which did not differ significantly among the treatments. Although it was not effective in reliably achieving cirrhosis, sweetening the alcohol solution with artificial sweeteners led to reasonable alcohol intakes with resultant hepatic fibrosis, and without the high carbohydrate intake which occurs when sucrose is used.

A sensitive method for the measurement of glycosylated plasma proteins using affinity chromatography.

We describe a simple, sensitive affinity technique for the routine measurement of glycosylated plasma proteins in clinical laboratories. The commercially available phenylboronic acid gel used for the chromatography has recently been marketed as a kit for this purpose (Glycogel Test Kit, Pierce Chemical Co). The manufacturers of this kit recommend loading 200 microliters neat plasma to each 1 ml gel column. This high loading is to enable the direct measurement of protein in the bound and unbound fractions at 280 nm. This loading is consistent with 10-15 mg protein being added per ml gel. Our results show that protein levels greater than 2 mg per ml gel overload the column. Therefore we used a modification of the more sensitive Bradford procedure to measure protein. The method discriminates between normals (6.29 +/- 1.87%) and diabetic patients (12.62 +/- 3.36%) and has good precision (CV 4-6%). The results obtained correlate with the colorimetric method using thiobarbituric acid (r = 0.70) and with glycosylated haemoglobin (r = 0.82).

An inexpensive, rapid and precise affinity chromatography method for the measurement of glycosylated haemoglobins.

We have assessed an affinity chromatography technique, using commercially available materials, for the estimation of total glycosylated haemoglobin in the routine clinical chemistry laboratory. The method gives good discrimination between normals (7.31 +/- 0.92%) and diabetics (12.70 +/- 2.88%) and has excellent precision (CV 1.5-2.0%). Labile glycosylated haemoglobin is normally removed as it is so variable. There is no significant correlation between labile glycosylated haemoglobin and blood glucose. Immediate analysis of incubated haemolysates is preferable to storage of haemolysates or erythrocytes. The affinity gel can be reused about 16 times, but oxidation must be reduced by keeping the gel at 4 degrees C in the dark when not in use. The cost of the gel is about 7p a test and 60 samples can be analysed in a working day. The method is not affected by the presence of up to 20% met-haemoglobin and should also give correct values for samples containing genetic variants of haemoglobin.

Tunneled right atrial catheter infection presenting as renal failure.

We report two cases of progressive renal failure secondary to membranoproliferative glomerulonephritis associated with subclinical septicemia from a tunneled right atrial catheter used for home parenteral nutrition administration. Although the occurrence of line infection and septicemia is a common complication of central venous catheters, a review of the literature reveals only one case report of renal failure secondary to an infected implanted central venous device. Both patients presented with azotemia and had biopsy-proven membranoproliferative glomerulonephritis, accompanied by leukocytoclastic vasculitis. In both cases, removal of the right atrial catheter and prolonged antibiotic therapy was effective in resolving the ongoing infection and resulted in marked improvement in renal function. A high index of suspicion for catheter sepsis should be maintained in patients with tunneled right atrial catheters presenting with subacute renal failure.

Cytoplasmic inclusions of Fabry's disease. Ultrastructural demonstration of their presence in urine sediment.

The diagnosis of Fabry's disease (angiokeratoma corporis diffusum universale) is usually confirmed by demonstrating typical cytoplasmic inclusions in a renal biopsy specimen, in conjunction with typical skin and ocular lesions and deficient alpha-galactosidase A activity in a male patient. Affected men are usually associated with carrier female family members. Electron microscopy of the urine sediment from two patients with Fabry's disease demonstrated typical laminated cytoplasmic inclusions within exfoliated epithelial cells. This method may be useful in the diagnosis of Fabry's disease and in screening of kin for hemizygotes and female carriers.

Primary ultrastructural diagnosis of cryoglobulinemic glomerulonephritis.

A 58-year-old woman suffering from epistasis was found to be hypertensive. Hypocomplementemia associated with normal serum creatinine level, erythrocytosis, microscopic hematuria, proteinuria, and normal serum immunoglobulin levels and immunoelectrophoresis results led to a renal biopsy. Light microscopy disclosed mesangial hypercellularity and accumulation of eosinophilic material in capillary loops and in the mesangium. Immunomicroscopy disclosed coarsely granular to globular deposition of IgG, IgM, and C3 in similar distribution. Electron microscopy demonstrated electron-dense mesangial and subendothelial deposits consisting of interlacing fibrillar bundles associated with infiltration of acute inflammatory cells characteristic of cryoglobulinemic glomerulonephritis. After the biopsy, the presence of circulating IgG-IgM cryoglobulins was documented. Substructure of the in vitro cryoprecipitate and the immune complexes in the kidney was very similar. These observations illustrate the importance of continued routine ultrastructural assessment in evaluation of renal biopsy material.

Strategies to prevent progression of renal disease.

Clinical studies show that several strategies can prevent or slow the progression of renal disease in patients with or without diabetes. This paper reviews current theories of how renal disease develops and progresses and what clinical studies indicate about how to prevent or slow the process.

Acute tubulointerstitial nephritis.

Between 1980 and 1988, 12 patients at the Cleveland Clinic had biopsy-proven acute tubulointerstitial nephritis. Etiologies of the disease included drugs, systemic illness, and idiopathic causes. Clinical features were nonspecific, and the diagnosis of acute tubulointerstitial nephritis was seldom entertained in these patients prior to biopsy. Seven patients had unrelated underlying renal disease. Treatment included discontinuation of the offending agent and/or a trial of steroids. All patients had final creatinine levels lower than at diagnosis. Because the condition is potentially reversible, this disease should be considered in all patients with new azotemia who do not exhibit prerenal factors, features typical of acute tubular necrosis, red blood cell casts heralding a glomerular process, or evidence of obstructive uropathy.