Induction of creatininase activity in chronic renal failure: timing of creatinine degradation and effect of antibiotics.

Creatinine degradation was prospectively studied in four healthy subjects and 35 patients with varying degrees of chronic renal failure by measuring creatininase activity in stool isolates. Patients were subdivided into those with serum creatinine above and below 6 mg/dL. Creatinine degradation in the former group of patients who had not taken antibiotics in the previous 3 months was significantly greater than the latter (64% v 26%; P < 0.001), which was similar to healthy controls. This degradation was abolished when antibiotics were added directly to the patient's stool during incubation (P < 0.002). In a subset of five patients, duodenal intubation demonstrated small bowel bacterial overgrowth associated with high concentrations of toxic methylamines generated there from and increased stool creatinine consumption. We conclude that retained creatinine in advanced chronic renal failure induces bacterial creatininase activity throughout the bowel, causing creatinine degradation and subsequent potential loss of creatinine to the creatinine pool. The modifying effects of antibiotics on creatinine degradation has important clinical implications for the interpretation of serum creatinine measurements in renal failure.

Biomodulation of the toxic and nutritional effects of small bowel bacterial overgrowth in end-stage kidney disease using freeze-dried Lactobacillus acidophilus.

Small bowel bacterial overgrowth (SBBO), well known to occur in end-stage kidney failure, is responsible for producing uremic toxins and contributing to the patient's decreased nutritional well-being. In this study, 8 hemodialysis patients were treated with a course of oral Lactobacillus acidophilus (LBA) in an attempt to alter this SBBO. LBA treatment was effective in lowering 2 compounds generated in vivo. Serum dimethylamine (DMA) levels dropped from 224 +/- 47 to 154 +/- 47 micrograms/dl at the end of LBA treatment (p < 0.001). Nitrosodimethylamine, a carcinogen, levels also decreased significantly from 178 +/- 67 (untreated) to 83 +/- 49 ng/kg (after LBA treatment). Patients nutritional status, assessed as serum albumin, body weight, caloric intake, midarm muscle area (MAMA) and appetite improved modestly, but not significantly. LBA changed small bowel pathobiology by modifying metabolic actions of SBBO, reducing in vivo generation of toxins and carcinogens and promoting nutrition with no adverse side effects.

N-nitrosodimethylamine blood levels in patients with chronic renal failure: modulation of levels by ethanol and ascorbic acid.

We measured levels of N-nitrosodimethylamine (NDMA) in peripheral blood from 13 fasting male patients, 30-74 years old, who had chronic renal failure, and in five healthy control subjects (four males and one female) 31-50 years old. In the patients, we found significant (P less than .01) levels of NDMA (mean +/- SD; 201 +/- 111 ng/kg of blood), which is known to be carcinogenic in animals. Five minutes after oral administration of ethanol (0.4 g/kg of body weight), all patients exhibited a significant (P less than .01) rise in blood NDMA levels (338 +/- 125 ng/kg), suggesting continuous endogenous formation of NDMA that was unmasked by ethanol's ability to inhibit first-pass hepatic metabolism of NDMA. In five of six patients, pretreatment with oral ascorbic acid resulted in a blunting, but not statistically significant, effect on maximum blood NDMA levels after consumption of ethanol. Mean levels were 340 +/- 100 ng/kg before treatment with ascorbic acid and 237 +/- 127 ng/kg during treatment. Ethanol administration unmasks increased gastrointestinal formation of NDMA in patients with chronic renal failure. Further studies are required to confirm a possible link between endogenous NDMA formation and the increased incidence of cancer in these patients.

Analysis of human blood for volatile N-nitrosamines by gas chromatography-chemiluminescence detection.

A method was developed to separate and measure trace levels of volatile N-nitrosamines (NAs) in human blood that either eliminated or accounted for in vitro artifactual formation of N-nitrosodimethylamine (NDMA) through the use of water blanks, added inhibitor (ascorbic acid) and added morpholine. The absolute minimum detectable limit was 8 pg; minimum level of reliable measurement was 0.05 microgram/kg for a 20-g blood specimen. Recovery of NDMA from blood was 93 +/- 5%. Coefficient of variation was 25%. Bloods from 242 people were analyzed for volatile NAs. NDMA was the only NA found. Positive specimens were presumptively confirmed by their non-detection after ultraviolet photolysis and/or mass spectrometry. This paper presents additional evidence that in vivo NA formation occurs.

Determination of body burden of uremic toxins.

The body burden of two uremic toxins, di- and trimethylamines, was determined in eight chronic renal failure patients, who received a renal transplant with immediate good function. The "pools" of these amines were calculated on the basis of serum concentration just prior to vascular anastomosis being established and compared to measured amines excreted over a period of five days following transplantation. The amount of DMA and TMA present intra-cellularly in eight muscle samples obtained from these patients was also studied. Both the measured to calculated ratios and intracellular to extracellular ratios of these amine concentrations suggests significant intracellular sequestration in end-stage renal disease. The possible effect on the intracellular milieu is discussed. Serum concentration of these uremic toxins under-estimates the total body burden.

Analysis for and intestinal metabolism of precursor nitroso compounds in normal subjects and in patients with chronic renal failure.

We have demonstrated that patients with chronic renal failure generate increased amounts of both dimethylamine and N-nitrosodimethylamine in the small bowel in association with aerobic and anaerobic bacterial overgrowth. The significance of these findings in relation to the reported increased incidence of cancer in patients with chronic renal failure has not yet been defined.

Evidence for generation of the precarcinogen nitrosodimethylamine in the small intestine in chronic renal failure.

We have previously reported raised concentrations of dimethylamine and also bacterial overgrowth in the small intestine in CRF. Evidence for in vivo NDMA formation in the same site in CRF is now presented. Gastroduodenal intubation was performed in 9 healthy volunteers and 7 patients with advanced chronic renal failure. Blood, gastric, and duodenal aspirates were analyzed for NDMA. NDMA levels in control and CRF patients for blood were normal, but for gastric aspirate they were 67 +/- 13 and 312 +/- 68 (P less than 0.001) and for duodenal aspirate they were 70 +/- 21 and 319 +/- 47 (P less than 0.001), respectively. The results of bacterial cultures confirmed small intestinal bacterial overgrowth. We thus demonstrated statistically significant differences between NDMA concentrations in the control subjects and patients for both gastric and duodenal aspirates. This suggests that there is increased intestinal generation of NDMA in uremia. The presence of this precarcinogen may be linked with the reported increase in the incidence of cancer in CRF.

Generation of dimethylnitrosamine in water purification systems. Detection in human blood samples during hemodialysis.

Dimethylnitrosamine (DMNA), a carcinogen, was detected at levels up to 32 micrograms/L in dialysate from five of 16 dialysis units surveyed. Blood drawn from patients at one of these units in which DMNA was raised in the dialysate showed a significant increase in the amount of DMNA in the patient's blood when predialysis levels were compared with 15-minute intradialysis levels. The presence of a mixed-bed deionizer without an antecedent carbon filter appeared to be necessary for DMNA production. These data suggest that DMNA is generated in certain water purification systems and may then diffuse into the patient's blood. Guidelines for deionizer-treated water should be revised to include an activated carbon filter.

Importance of aliphatic amines in uremia.

Five main aspects were addressed: 1)The demonstration that creatinine is an endogenous precursor of dimethylamine (DMA) in chronic renal failure. 2) The size of the body amine pool measured in transplant patients suggests sequestration in some intracellular compartment. This illustrates the possible error in directly relating serum concentrations to neurological toxicity. 3) Bacterial overgrowth and increased generation of duodenal DMA in the small intestine becomes apparent at a serum creatinine above 8 mg/dl. Two cases show that bacterial overgrowth preceded the increased duodenal DMA. 4)Clinical toxicity is demonstrated by i) correlation of abnormal neurobehavioral parameters with serum amine levels, and ii) by improvement with administration of nonabsorbable broad spectrum antibiotics. Results with adsorption agents are inconclusive. 5) Preliminary tests of behavior modification in a rat model by direct instillation of amines into the brain are positive for TMA but negative for DMA, but no DMA entry into brain cells is demonstrated in the latter. The generation of aliphatic amines represents only one part of a spectrum of alteration induced by proximal intestinal bacterial enzyme action that occurs in renal failure. It is possible that some bacterial activity is beneficial and that the net clinical result is a balance between the "good" and the "evil" bacterial effects.

Bacterial populations of the small intestine in uremia.

The small intestinal bacterial flora of 15 patients with chronic renal insufficiency was compared with that of subjects with blind loop synDROME. 9 patients were on regular hemodialysis with high protein intake and 6 (serum creatinine 7.5 to 12.5 mg/dl) were maintained on low protein diet. The chronic renal patients harbored a greatly increased microbial flora of both anaerobes and aerobes in the duodenum and jejunum, quantitatively comparable to those in blind loop subjects. The composition did not differ significantly in the two groups. Some organisms may have the potential to metabolize substrates which reach the intestinal lumen from the diet and bile, and perhaps to generate toxic metabolites that could contribute to uremic toxicity or malabsorption.

Biochemical profile of uremic breath.

We attempted to define the substances that contribute to the characteristic "uremic breath" of patients with end-stage renal disease. Breath samples from nine patients underwent direct analysis before and after hemodialysis with use of gas chromatography and confirmation by mass spectrometry, and indirectly assessment by an organoleptic panel. Concentrations of secondary and tertiary amines, dimethylamine and trimethylamine were increased, with subsequent reduction after hemodialysis (dimethylamine from 2.00 +/- 0.19 [S.E.M.] to 0.88 +/- 0.12 microng per 30 minutes, P less than 0.001, and trimethylamine from 0.79 +/- 0.22 to 0.44 +/- 0.15 microng per 30 minutes, P less than 0.003). Treatment with nonabsorbable antibiotics in two patients reduced both serum and breath amine levels without dialysis. Loss of nitrogen via the breath was not quantitatively important. We conclude that uremic breath reflects the systemic accumulation of potentially toxic volatile metabolites, among which dimethylamine and trimethylamine have been positively identified and correlated with the classic fishy odor.

Toxicity of aliphatic amines in uremia.

Fifty-three samples in 26 patients were analyzed for aliphatic amines (DMA and TMA), and the levels correlated with 2 neurophysiological tests, choice reaction time (CRT), and electroencephalogram (EEG). A significant correlation was found between TMA and CRT and EEG (p less than 0.001 and 0.003, respectively) and between DMA and CRT (p less than 0.01). These amines reflect part of the spectrum of toxic compounds which accumulate in uremia. Dissociation of neurophysiological functions may be helpful in evaluating various classes of potentially toxic compounds found in renal failure, as exemplified by short-chain aliphatic amines.

Amine metabolism and the small bowel in uraemia.

Intestinal intubation was carried out in 21 subjects: 9 with end-stage renal failure, 2 with early renal insufficiency, 7 untreated patients with blind-loop syndrome, and 3 normal volunteers. All 9 patients with uraemia had significantly raised duodenal dimethylamine (D.M.A.) concentrations compared with the other groups tested. Alteration of the intestinal bacterial flora with antibiotics markedly reduced serum D.M.A. and trimethylamine concentrations and modified the symptoms. Potentially toxic metabolites in the small bowel might have significant nutritional and toxic sequelae in uraemia and these findings suggest that current therapeutic approaches to the treatment of end-stage kidney disease should be re-examined in relation to bowel flora.