Earle W. Holmes

Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage

Objective: Only 30% of alcoholics develop cirrhosis, suggesting that the development of alcohol-induced liver injury requires one or more additional factors. Animal studies have shown that gut-derived endotoxin is one such factor. Because increased intestinal permeability has been shown to cause endotoxemia, we hypothesized that increased gastrointestinal permeability contributes to the pathogenesis of alcoholic liver disease. This study aimed to measure gastroduodenal and intestinal permeability in alcoholics with and without chronic liver disease and in nonalcoholic subjects with chronic liver disease. Methods: Gastroduodenal permeability was assessed by measurement of urinary excretion of sucrose after oral administration. Intestinal permeability was assessed by measurement of urinary lactulose and mannitol after oral administration of these sugars. Results: Alcoholics with no liver disease showed a small but significant increase in sucrose excretion. Alcoholics with chronic liver disease demonstrated a marked and highly significant increase in urinary sucrose excretion relative to the controls, to the alcoholics with no liver disease, and to the nonalcoholics with liver disease. Alcoholics with chronic liver disease demonstrated a marked and highly significant increase in both lactulose absorption and in the urinary lactulose/mannitol ratio (alcoholics 0.703 vs controls 0.019, p= 0.01). In contrast, alcoholics with no liver disease and nonalcoholics with liver disease showed normal lactulose absorption and normal lactulose/mannitol ratio. Conclusion: Because only the alcoholics with chronic liver disease had increased intestinal permeability, we conclude that a “leaky” gut may be a necessary cofactor for the development of chronic liver injury in heavy drinkers.

Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography

The status of the oxidative metabolism of L-tryptophan is usually evaluated by the determination of tryptophan metabolites in serum or urine and/or the activities of various oxidative enzymes in tissues. I have developed assays for serum kynurenine and hepatic tryptophan dioxygenase (TDO) activity based on the determination of kynurenine (KYN) by isocratic, reverse phase HPLC with spectrophotometric detection at 365 nm. Sample pretreatment prior to HPLC requires little more than perchloric acid precipitation of serum or a TDO incubation mixture. The analytical recovery for the serum assay was 101 +/- 2%, while the run-to-run coefficient of variation at normal KYN levels was approximately 8%. Serum KYN levels in 40 apparently healthy fasting humans were normally distributed and ranged from 0.27 to 0.69 microgram/ml (mean +/- SD: 0.47 +/- 0.1). Serum KYN in predialysis specimens from a group of 20 patients with chronic renal failure demonstrated a highly significant increase (mean +/- SD: 0.83 +/- 0.35 microgram/ml; P less than 0.001) as compared to the reference population. It is possible that such an increase might contribute to the pathophysiology of the uremic state. The analytical recovery of KYN from TDO incubation mixtures was approximately 90%. There was no evidence for the onward metabolism of KYN during the assay of whole liver homogenates. The mean (+/- SD) TDO activity of rat liver homogenates preincubated with ascorbate and hematin was 2.3 +/- 0.8 mumol/h/g wet wt (30 degrees C). The sensitivity, specificity, and convenience of these two methods suggest that they are suitable for routine use in the investigation of the biology and pathology of oxidative tryptophan metabolism.

Glutathione content of colonic mucosa : Evidence for oxidative damage in active ulcerative colitis

Oxidative stress appears to play a role in thetissue damage of active ulcerative colitis, and it hasbeen suggested that a defect in mucosal antioxidantdefenses is a etiological factor in the disease. This study was undertaken to investigate themucosal content and oxidation state of glutathione inulcerative colitis in the active and inactive states andto examine the relationship between glutathione content and disease activity in this patientpopulation. Endoscopic biopsies of colon mucosa werecollected from normal subjects, from macroscopicallynormal tissue of patients with inactive and active ulcerative colitis, and from inflamed tissue ofpatients with active ulcerative colitis. The mucosalcontents of GSH and GSSG were determined by liquidchromatography. We found no significant differences in tissue contents of reduced glutathione amongthe four groups. The median tissue level of oxidizedglutathione in inflamed mucosa from patients with activeulcerative colitis was increased 1.7-fold (P = 0.017)over that of patients with inactive disease. Theoxidized glutathione content of the mucosa also showedsignificant positive correlations with clinical andhistological indices of disease severity among ulcerative colitis patients. Inconclusion, a change in the redox status of mucosalglutathione is associated with inflammation and diseaseactivity in ulcerative colitis. This change appears tobe a consequence of inflammation rather than apathogenic factor for the disease.

Gas chromatographic method for detection of urinary sucralose: application to the assessment of intestinal permeability.

We developed a capillary column gas chromatography (CCGC) method for the measurement of urinary sucralose (S) and three other sugar probes including, sucrose, lactulose (L) and mannitol (M) for use in in vivo studies of intestinal permeability. We compared the capillary method with a packed column gas chromatography (PCGC) method. We also investigated a possible role for sucralose as a probe for the measurement of whole gut permeability. Sample preparation was rapid and simple. The above four sugars were detected precisely, without interference. We measured intestinal permeability using 5- and 24-h urine collections in 14 healthy volunteers. The metabolism of sugars was evaluated by incubating the intestinal bacteria with an iso-osmolar mixture of mannitol, lactulose and sucralose at 37 degrees C for 19 h. Sugar concentrations and the pH of the mixture were monitored. The use of the CCGC method improved the detection of sucralose as compared to PCGC. The average coefficient of variation decreased from 15% to 4%. It also increased the sensitivity of detection by 200-2000-fold. The GC assay was linear between sucralose concentrations of 0.2 and 40 g/l (r=1.000). Intestinal bacteria metabolized lactulose and acidified the media but did not metabolize sucralose or mannitol. The new method for the measurement of urinary sucralose permits the simultaneous quantitation of sucrose, mannitol and lactulose, and is rapid, simple, sensitive, accurate and reproducible. Because neither S nor M is metabolized by intestinal bacteria, and because only a tiny fraction of either sugar is absorbed, this pair of sugar probes appears to be available for absorption throughout the GI tract. Thus, the 24-h urinary concentrations of S and M, or the urinary S/M ratio following an oral dose of a sugar mixture, might be good markers for whole gut permeability.

Renal and hepatic output of glutathione in plasma and whole blood

Measurement of glutathione (GSH) output by the rat kidney and liver demonstrated a substantial net release into red cells across both tissues. The results suggest important roles for kidney and liver in the maintenance of GSH concentrations in red cells and a significant role for the red cell in the interorgan transport of GSH.

Analytic bias among certified methods for the measurement of hemoglobin A1c: a cause for concern?

We studied the magnitude, significance, and origin of an analytic bias that emerged between our point-of-care (POC) and our central laboratory (CL) methods for the measurement of hemoglobin A1c (HbA1c) and evaluated the analytic accuracy of 7 commonly used HbA1c methods relative to the National Glycohemoglobin Standardization Program (NGSP) reference method. The POC and CL methods were compared by split-sample analysis of clinical specimens and time series analyses of the HbA1c results reported for a 33-month period. The relative accuracies of 7 HbA1c methods were evaluated using College of American Pathologists proficiency survey results. Long-term drifts in the CL- and POC-analyzed test results caused the median intermethod bias [(POC result)-(CL result)] to increase from -0.4% to -0.9% HbA1c. Systematic biases, drifts in analytic performance over time, and intermethod variability were frequently observed among the 7 NGSP-certified HbA1c methods. Intermethod variability is a potential source of inaccuracy whenever HbA1c results are interpreted relative to universal, fixed, clinical decision thresholds.

Analytical variability among methods for the measurement of 25-hydroxyvitamin D: still adding to the noise.

OBJECTIVES To compare total 25-hydroxyvitamin D [25(OH) D] results measured by 3 direct immunoassays, including the previous version of the DiaSorin Liaison2 assay and the current versions of the Siemens Centaur2 and the Abbott Architect assays, with results measured in serum extracts by liquid chromatography/tandem mass spectrometry (LC/MS) and radioimmunoassay (RIA). METHODS Our study sample consisted of 163 consecutive clinical specimens submitted to our laboratory for 25(OH)D testing. RESULTS Regression and bias analyses of the data revealed that results measured by the 3 direct immunoassay methods had high degrees of random variability and bias relative to the results determined by LC/MS and RIA. The relative biases between results measured by the direct assays and the comparison methods exceeded a recommended criterion for the total allowable error of a 25(OH)D test in as many as 48% of our clinical specimens. Of the subjects in our study sample, 33, 37, 30, 45, and 71 were classified as vitamin D deficient based on results determined by LC/MS, RIA, Liaison2, Architect, and Centaur2, respectively. CONCLUSIONS Intermethod variability in 25(OH)D assays continues to limit our progress toward the establishment of reference values for 25(OH)D in health and our efforts to gain a better understanding of the role of vitamin D insufficiency as a risk factor for disease.

Tryptophan distribution and metabolism in experimental chronic renal insufficiency

Several aspects of tryptophan distribution and metabolism in chronic renal insufficiency (CRI) were investigated in a rat model prepared by partial nephrectomy. Partially nephrectomized (pNx) rats with a moderate degree of CRI demonstrated a 40% decrease in plasma total tryptophan concentration between 5 and 11 weeks after the acute reduction of renal functional mass. This decrease was accompanied by hypoalbuminemia, polyuria, albuminuria, and tryptophanuria. After 5 weeks of sustained plasma total tryptophan deficiency (from Week 6 to Week 11), the plasma free tryptophan concentration, the plasma concentrations of large neutral amino acids, and the tryptophan levels in red cells, liver, and kidney of the pNx rats were similar to those of the controls. However, evidence for abnormal brain tryptophan metabolism in pNx rats after 11 weeks of CRI included 16% reductions of tryptophan levels in the midbrain and pons and 65% increases in the serotonin contents of the hypothalamus and medulla. Monoamine oxidase activities in hypothalamus and cerebellum of pNx rats were the same as those of the controls. These studies indicate that tryptophanuria is an important factor in the development of the plasma tryptophan deficiency in the pNx model. In addition, the results support the hypothesis that regional abnormalities in tryptophan metabolism contribute to the neurological and neuroendocrine dysfunction of CRI.

Chronic fluoxetine reduces autonomic control of cardiac rhythms in rats with congestive heart failure

Up to 40% of patients with heart failure develop depression, and depression is an independent risk factor for cardiovascular mortality in this patient population. Consequently, increasing numbers of patients with heart failure are treated with antidepressants. Selective serotonin reuptake inhibitors are typically the antidepressant of choice since this drug class has limited cardiovascular toxicity. However, little is known about the effects of selective serotonin reuptake inhibitors on autonomic cardiac regulation in congestive heart failure (CHF). Here, indexes of cardiac autonomic control were evaluated before and during chronic fluoxetine (FLX) treatment (20 mg·kg(-1)·day(-1), 5 wk) in rats that developed CHF after coronary artery ligation. FLX reduced the low-frequency (LF) component of heart rate variability (HRV; P < 0.01) as well as the sympathetic contribution to LF HRV (P < 0.01) in both CHF and sham-operated rats. Both FLX and CHF reduced high-frequency HRV (P < 0.01). Spontaneous baroreflex gain was decreased in CHF rats 8 wk after ligation (P < 0.01). Cross-spectral coherence between the interbeat interval and mean arterial pressure was reduced in the LF domain 3 wk after ligation in CHF rats (P < 0.01) and was further reduced after chronic FLX treatment (P < 0.01). Plasma catecholamines and LF blood pressure variability were not affected by FLX. Chronotropic responses to both efferent vagal nerve stimulation and isoproterenol administration were reduced in CHF rats and by FLX (P < 0.01), whereas inotropic responses to isoproterenol were reduced only in CHF rats (P < 0.01). These data indicate that chronic FLX reduces the responsiveness to autonomic output controlling cardiac rhythm and may further compromise autonomic regulation of cardiac function in CHF.

Determination of Ultrafilterable Prolactin: Elimination of Macroprolactin Interference With a Monomeric Prolactin-Selective Sample Pretreatment

CONTEXT Macroprolactin (macroPRL), present in as many as 25% of serum specimens with elevated serum prolactin concentrations, can cause apparent hyperprolactinemia in the absence of clinical features and lead to unnecessary clinical, laboratory, and neuroradiological workups. OBJECTIVE To develop an ultrafiltration method that eliminates macroPRL interference from PRL immunoassays. DESIGN The method involves centrifugation of undiluted serum in a Centricon-100 filter device followed by a PRL assay of the serum ultrafiltrate. RESULTS Ultrafiltrates prepared by this technique are devoid of gamma globulins and contain (mean +/- SE) 19% +/- 7% of the albumin concentration of the original serum. These ultrafiltrates contain 85% +/- 7% of the total PRL immunoreactivity of serum spiked with 23 kd recombinant human prolactin (rHuPRL) and less than 2% of the 50 kd big PRL (bPRL) of whole serum. The fractional recovery of ultrafilterable PRL (uPRL) from serum samples of 54 female patients was 0.78 (confidence interval 0.73-0.83) of the total. The run-to-run coefficient of variation of the uPRL assay was 4.3%. The uPRL concentration (mean +/- SD) in a group of healthy female controls was 8.0 +/- 3.1 ng/mL. CONCLUSIONS Ultrafiltration is a rapid and simple method for eliminating analytical interference by macroPRL. Ultrafiltrates can be analyzed by most, if not all, currently available PRL immunoassays and represent a practical and precise alternative to gel filtration chromatography for the estimation of the monomeric prolactin concentration of serum.