Hepatic stellate cell activation in nonalcoholic steatohepatitis and fatty liver.

Factors associated with the development of fibrosis in nonalcoholic steatohepatitis (NASH) are largely unknown, although an association with increased hepatic iron has been suggested. Hepatic stellate cells are the principal collagen-producing cells in many liver diseases and when activated express alpha-smooth muscle actin (alpha-SMA). Hepatic stellate cell activation and association with fibrosis, necroinflammatory activity, steatosis, and stainable iron in 60 cases of NASH and 16 cases of steatosis were evaluated. All 76 patients were obese or had other risk factors for NASH. All biopsy specimens were stained for alpha-smooth muscle actin to evaluate the pattern of hepatic stellate cell activation and were evaluated for inflammatory activity (0 to 3), fibrosis (0 to 4), and stainable iron stores (0 to 4). The zonal location of activated stellate cells was recorded, and the degree of activation was graded as high-grade or low-grade based on the percentage of lobular alpha-SMA+ cells. Activated stellate cells were identified in the hepatic lobule in 74 of 76 biopsy specimens and graded as low-grade in 26 and high-grade in 48. Zone 3 was involved in 72 of 74 positive cases, and in 33 cases, the activated stellate cells were preferentially located in zone 3. The degree of stellate cell activation correlated with fibrosis but not with inflammatory activity, severity of steatosis, or stainable iron. In most cases, the degree of stellate cell activation paralleled the degree of hepatic fibrosis, but in 25 cases, the degree of hepatic stellate cell activation was greater than expected, raising the question of whether such patients are at risk for disease progression.

The significance of microabscesses in liver transplant biopsies: a clinicopathological study.

Parenchymal microabscesses (MA) in liver transplant biopsies are frequently associated with cytomegalovirus (CMV) infection. However, other potential causes of MA have not been fully investigated. We studied additional etiologies for MA via histological evaluation and clinicopathological correlation. Three hundred seventy-two liver transplant biopsies from 97 patients (from 1991 to 1997) were reviewed and stained immunohistochemically for CMV. Numerous histological features were evaluated including size and number of MA, lobular and portal inflammation, and cholestasis. Medical records were reviewed for radiographic, laboratory, and other clinical data from the time of biopsy. The chi2 or Fisher's Exact test and ANOVA with adjusted multiple comparisons were used to determine statistical significance. Sixty-two of 372 biopsies (17%) from 43 patients contained MA. Biopsies were obtained between 4 days and 2.3 years posttransplant (median, 14 days). Nineteen percent of biopsies had CMV infection at the time of biopsy; 27% were associated with other bacterial, viral, or fungal infections; 10% had graft ischemia; 15% had biliary obstruction/cholangitis; 3% had a combination of ischemia and sepsis; and no explanation was found in 26% of biopsies. Numerous MA within a biopsy (>9) correlated with CMV infection (P <.005); no other histological features, including size of MA, correlated with the etiology of MA. Overall, 43 of 97 (44%) liver transplantation patients at our institution had biopsies demonstrating MA at some point in their posttransplantation course. CMV infection appears responsible for only a minority of cases. MA, although nonspecific, are an important histological finding in numerous conditions that may have a significant impact on both graft survival and overall patient morbidity.

Experience with neoral versus sandimmune in primary liver transplant recipients.

We compared results using Neoral versus Sandimmune, each in combination with steroid and azathioprine immunosuppression, in primary liver transplantation recipients. There were 15 patients in each group with similar demographic distributions. Intravenous cyclosporine was stopped at 4.3 +/- 1.9 days in the Neoral group vs 7.8 +/- 4.9 days in the Sandimmune group. (P < 0.025). Cyclosporine levels in the first 10 days were higher (mean 306 ng/ml vs 231 ng/ml) in the Neoral group than the Sandimmune group (P < 0.05). The Neoral dose was less than the Sandimmune dose (mean 5.5 ng/kg per day vs 7.9 ng/kg per day) to achieve these levels in that time period (P < 0.05). Two patients (13%) experienced three episodes of biopsy-proven rejection in the Neoral group compared to nine patients (60%) with 12 episodes of rejection in the Sandimmune group (P < 0.025). Incidences of neurological and renal complications were similar between the groups. Infections requiring treatment were also similar. Liver function, renal function, and marrow function, evaluated at days 7, 14, 21, 28, and 2, 4, 6, and 12 months post-transplant, were not different between the groups. In summary, shorter use of intravenous cyclosporine and quicker stabilization of trough cyclosporine levels was achieved with Neoral than with Sandimmune. In the early post-transplant period, higher levels with lower doses were achieved with Neoral than with Sandimmune. In our experience, the incidence of rejection was lower with Neoral than with Sandimmune. There were similar lengths of hospitalization, mortality, adverse events, retransplantation, and similar liver, renal, and marrow function up to 1 year post-transplantation. Because of this experience, we continued to use Neoral in a total of 59 primary liver transplant recipients. We have not used intravenous cyclosporine in the last 44 patients. Follow-up was a mean of 11.4 months, ranging from 1 to 27 months. The incidence of rejection was 24% in these 59 patients compared to our historical experience of 70% using Sandimmune.

International trends in sales of inhaled fenoterol, all inhaled beta-agonists, and asthma mortality, 1970-1992.

To evaluate the hypothesis that fenoterol or all inhaled beta-agonists caused an epidemic of asthma mortality in New Zealand from the late 1970s to the mid-1980s, we examined trends from 1970 to 1992 in per capita sales of inhaled fenoterol, inhaled beta-agonists, and asthma mortality in New Zealand and nine other countries that marketed fenoterol. During the last two decades, there has been a large and widespread increase in sales of inhaled beta-agonists, including fenoterol. Asthma mortality in most countries, however, has been relatively stable. Only New Zealand experienced an epidemic of asthma mortality. In addition, sales rates of fenoterol similar in magnitude to those in New Zealand near the peak of the epidemic also occurred in Belgium, Austria, and Germany, while asthma mortality in these countries remained low. Also, sales rates of all beta-agonists in Australia were similar to those in New Zealand, but no epidemic of asthma mortality occurred in Australia. Therefore, the difference between asthma mortality rates in New Zealand and other countries is not explained by differences in per capita sales of fenoterol or all beta-agonists. Within New Zealand, the beginning and end of the epidemic correlated with a rise and fall in sales of all beta-agonists, including fenoterol. From 1980 to 1989, however, sales of fenoterol and all beta-agonists doubled in New Zealand while asthma mortality declined by 40%. International data on medication sales and asthma mortality, therefore, do not point to a relation between asthma mortality and beta-agonists in general nor fenoterol in particular.

Nashville experience with liver transplantation in Veterans Administration patients.

In this study, we compared results of 28 liver transplants performed in 25 patients referred through the Veterans Administration with 82 transplants performed in 75 nonveteran patients. The evaluation and follow-up care was provided by the same team of physicians and nurses and the transplant procedure performed in the same hospital for both patient groups. There was a significantly greater incidence of hepatitis C and/or previous alcohol abuse in veteran compared with non-VA patients [23/25 (92%) vs 29/75 (39%); P < 0.05] and a greater incidence of native portal vein thrombosis [6/25 (24%) vs 2/75 (2.6%); P < 0.01], but no difference in Child's-Pugh score (10.8 vs 9.9) or UNOS listing status (mean status 2.7 vs 2.8). The increased incidence of native portal vein thrombosis did not appear to be solely related to previous alcohol abuse or hepatitis C, since only 1 of 29 (3.4%) non-VA patients with these etiologies had this finding. There was no difference in patient or graft survival between the VA and non-VA groups with overall actuarial 6-, 12-, and 18-month patient survival of 86, 84, and 83% and graft survival of 80, 78, and 77%. There was no difference in major complication rates but there was a significantly longer average hospital stay (27 +/- 31 vs 18 +/- 12 days; P < 0.05) in the VA compared with non-VA group. One patient with native portal vein thrombosis in the non-VA group developed portal vein thrombosis in the postoperative period. There was no documented recidivism in any patient with a history of prior substance abuse in either group. This study confirms that veteran patients have a higher incidence of hepatitis C and previous alcohol abuse as causes of liver disease, have a higher incidence of native portal vein thrombosis, and have longer mean hospital stays, but experience the same survival in the first 18 months compared with nonveteran patients.

Individual nonsteroidal antiinflammatory drugs and other risk factors for upper gastrointestinal bleeding and perforation.

We conducted a nested case-control study of 1,377 cases of upper gastrointestinal bleeding or perforation (UGIB) and 10,000 controls to evaluate the association of individual nonsteroidal antiinflammatory drugs (NSAIDs), utilization characteristics, and other risk factors for these conditions. Age was the strongest risk factor for UGIB. Male gender, history of complicated peptide ulcer disease, and current use of steroids were also risk factors for UGIB. The adjusted odds ratio (OR) for current NSAID use was 4.3 [95% confidence interval (CI) = 3.7-5.0]. The ORs for current NSAID use were similar for fatal cases and for the gastric, duodenal, prepyloric, and multiple sites of lesion, but the OR was substantially increased for perforations (OR = 16.9;95% CI = 9.1-31.5). Women age 80 years and older experienced the greater effect of NSAID use. Current users of multiple NSAIDs and recent switchers showed ORs of 9.0 and 6.2, respectively. Ibuprofen showed the lowest OR and diflunisal, the highest. ORs for low, medium, and high NSAID daily dose were 2.9, 4.2, and 5.8, respectively. This trend was present among new, short-term, and long-term users. Simultaneous use of multiple NSAIDs as well as use of a single individual NSAID at high doses greatly increases the risk of complicated peptic ulcer disease.

Nonsteroidal anti-inflammatory drugs and the risk of hospitalization for acute renal failure.

BACKGROUND: Few studies have evaluated the association between use of nonsteroidal anti-inflammatory drugs (NSAIDs) and the risk of idiopathic acute renal failure (ARF) in the general population. METHODS: Population-based case-control study among persons in the Canadian province of Saskatchewan who received at least 1 NSAID prescription between January 1, 1982, and December 31, 1986. Health department databases were used for case detection, as the sampling frame for selecting controls (n = 1997), and as the primary source of information on drug use and comorbidity. A total of 306 hospital records were reviewed. Twenty-eight patients who were hospitalized fulfilled the diagnostic criteria for ARF. RESULTS: The incidence rate of hospitalization for ARF among the general population not exposed to NSAIDs was 2 per 100000 person-years. Current exposure to NSAIDs, acetylsalicylic acid and other nephrotoxic drugs, male gender, increasing age, cardiovascular comorbidity, and recent hospitalization for disorders other than renal were found to be independent risk factors for ARF. Current NSAID users had an adjusted odds ratio for ARF of 4.1 (95% confidence interval, 1.5-10.8). The risk of ARF was especially high during the first month of use (odds ratio, 8.5). For prescribed dose, we found that users of high daily doses of NSAIDs experienced an odds ratio of 9.8 for ARF. CONCLUSIONS: In the general population, hospitalizations for ARF were found to be a rare condition. The 4-fold increase in risk associated with NSAID use was dose-dependent and occurred especially during the first month of therapy. Concurrent comedication with other potentially nephrotoxic agents should be prescribed with care, especially in the elderly.

Outcomes analysis for 50 liver transplant recipients: the Vanderbilt experience.

Healthcare reform has mandated scrutiny of the fiscal aspects of patient care as well as medical outcomes. Therefore, we reviewed our experience with 50 liver transplant recipients from a multidisciplinary collaborative transplant team. From February 1991 to July 1994, of 175 patients referred, 75 were formally evaluated for transplantation; 56 (76%) of these patients were accepted for transplantation; 50 patients underwent 53 transplants. Operative mortality of 6 per cent, retransplantation rate of 6 per cent, 6-month actuarial survival of 88 per cent, 1-year survival of 86 per cent, and the 2 and 3-year survival of 83 per cent were unchanged over time. Quality of life evaluated by the Karnofsky Performance Status was a mean of 55 pretransplant, 72 at 3 months, 79 at 6 months, 84 at 1 year, 88 at 2 years, and 95 at 3 years, demonstrating improved general health and functional rehabilitation after transplantation. Psychosocial Adjustment to Illness Scale scores demonstrated significant improvement following transplantation, improving most dramatically in the vocation environment, domestic environment, and sexual relationship domains. Postoperative length of stay has declined with an average of 28 days in 1991, 22 days in 1992, 19 days in 1993, and 14 days in 1994. Average total hospital, organ procurement, and physician charges for the transplantation hospitalization was $165,000. Average 91-92 hospital charges were $154,000 and were reduced in 93-95 to $103,000 (P < .05). We found that charges and length of stay decreased over time, while the outcome and quality of patient care was maintained. We believe the collaborative practice, case management, and revised patient care protocols are responsible.

Positive predictive value of ICD-9 codes in the identification of cases of complicated peptic ulcer disease in the Saskatchewan hospital automated database.

We examined the positive predictive value of the International Classification of Diseases, 9th revision (ICD-9), codes used to identify cases of complicated peptic ulcer disease from the Saskatchewan Hospital automated database. Nonspecific and site- and lesion-specific codes were used in case detection to increase the sensitivity of the initial computer search. Review of the hospital records of 1,762 potential cases resulted in 1,375 confirmed cases. The positive predictive value of site and lesion-specific codes was about 90% and was about 70% for the nonspecific codes. Almost 50% of cases, however, would have been missed if the nonspecific codes had not been used.

Pruritus of chronic cholestasis.

Pruritus is a challenging clinical problem which often complicates chronic cholestatic liver disease. For practical purposes, cholestasis may be defined as impaired hepatocellular secretion of bile and is a feature of a wide variety of liver diseases. Cholestasis is usually suspected clinically when a patient presenting with jaundice or pruritus is found to have an elevation in serum alkaline phosphatase activity disproportionate to increases in serum aminotransferase levels. Early imaging by ultrasonography, computerized tomography, or cholangiography is important to address the possibility of remediable biliary tract obstruction. The majority of patients who develop problematic pruritus due to chronic cholestasis will have one of several diseases: primary biliary cirrhosis, primary sclerosing cholangitis, drug-induced cholestasis, autoimmune chronic active hepatitis, or alcoholic liver disease. Specific aetiological diagnosis is usually possible when history and physical examination are complemented, as appropriate, by serological testing, hepatobiliary imaging, and liver biopsy. This review does not address issues in diagnosis, but concentrates upon the management of pruritus, a potentially disabling complication of prolonged cholestasis.

Rupture and hemorrhage of hepatic focal nodular hyperplasia.

Although adenoma and focal nodular hyperplasia (FNH) are both benign liver lesions, adenomas are associated with a risk of rupture and malignant degeneration. This had led to the general recommendation of resection of adenomas. However, FNH rarely ruptures or becomes malignant, and a nonoperative approach has been adopted by most hepatobiliary centers when the diagnosis of FNH can be made with reasonable certainty. There are only two previous reports of rupture of FNH in the English literature; we present a third case of FNH with spontaneous rupture and hemorrhage. An 18-year-old healthy Caucasian woman presented with sudden onset of severe RUQ pain. She had never been pregnant, nor used oral contraceptive agents, and had not sustained major trauma. Her abdominal exam revealed RUQ tenderness on palpation. Hepatic biochemical tests, CBC, and coagulation tests were normal. Her hematocrit of 44% fell to 31% over 48 hours. CT scan revealed right anterior lobe and left medial segment hypodense liver lesions (4-5 cm) as well as hemoperitoneum and angiography revealed hypervascular lesions. At laparotomy, two tan fibrous subcapsular masses were excised. Pathology showed a central stellate scar in both lesions with several nodules surrounding the central scar on microscopic section, characteristic of FNH. There was evidence of hemorrhage in one lesion. Significant symptoms are an indication for resection of FNH lesions. However, most patients with FNH are asymptomatic and have a normal physical exam. The natural history of these lesions is enigmatic, and the indications for surgery are still evolving. This report emphasizes that a small risk of rupture clearly exists.(ABSTRACT TRUNCATED AT 250 WORDS)

Kupffer cell stimulation with Corynebacterium parvum reduces some cytochrome P450-dependent activities and diminishes acetaminophen and carbon tetrachloride-induced liver injury in the rat.

Chemical activation of Kupffer cells in vivo by vitamin A or latex beads is associated with a worsening of hepatic injury induced by the P450-dependent hepatotoxins acetaminophen (ACET) and carbon tetrachloride (CCl4) and by the P450-independent toxin galactosamine (GLN). Immunostimulants such as Corynebacterium parvum (CP) also activate Kupffer cells, but do so while prompting release of soluble mediators which depress microsomal oxidative activities in cultured hepatocytes. Therefore, we sought to characterize the effects of CP on hepatic injury in vivo due to ACET and CCl4 while employing GLN as a control. Hepatic microsomal oxidative activity and glutathione (GSH) disposition were examined since each influences susceptibility to injury from ACET or CCl4. Rats were given CP 28 mg/kg i.v. 5 days before challenge with hepatotoxicant. Hepatic injury was assessed 24 hr after hepatotoxicant administration by measurement of serum alanine aminotransferase (ALT) activity and review of histological sections. Livers from parallel groups of rats were used to prepare microsomal and cytosolic fractions, to measure tissue GSH, or for perfusion to assess GSH efflux. Significant reductions in injury due to ACET or CCl4 were observed while injury due to GLN was potentiated. Serum ALT levels after ACET were 3000 +/- 620 in controls vs 170 +/- 45 IU/liter in the CP-treated group and ALT levels after CCl4 were 3100 +/- 500 in controls vs 1700 + 450 IU/liter in the CP-treated group. In contrast, serum ALT levels after GLN were 920 +/- 230 in controls vs 1700 +/- 370 in the CP-treated group. Patterns of hepatic injury observed on histological sections were those characteristic for each toxin and the severity of injury correlated well with alterations in serum ALT levels for each agent. Hepatic microsomal fractions from rats pretreated with CP showed significantly diminished total cytochrome P450 content as well as reduced activity for two P450IIE1 substrates, p-nitrophenol and 7-ethoxycoumarin. While sinusoidal efflux of GSH increased by 40% in rats pretreated with CP and cytosolic glutathione-S-transferase activity fell slightly, tissue GSH levels were unaffected. These data demonstrate that CP decreases microsomal cytochrome P450 content, reduces biotransformation of two P450IIE1 substrates, and diminishes ACET- and CCl4-induced hepatic injury. In contrast, hepatic injury due to the P450-independent toxin GLN was enhanced. Thus, chemical and immune stimulation of Kupffer cells may result in divergent effects on susceptibility to injury from individual hepatotoxins.

Ethanol-associated alterations in the kinetics of putrescine uptake and metabolism by the regenerating liver.

Biosynthesis of the polyamines, putrescine, spermidine, and spermine is required for DNA synthesis and liver regeneration after partial hepatectomy. We have previously reported that chronic ethanol consumption impairs polyamine synthesis and significantly retards liver regeneration after partial hepatectomy. In those studies, supplementation with putrescine restored hepatic DNA synthesis in ethanol-fed rats but exerted no effect in pair-fed controls. These differences in the response to putrescine treatment may have resulted from ethanol-associated differences in hepatic uptake, release, or metabolism of putrescine. To resolve these issues and define more completely how putrescine treatment affects DNA synthesis, we now assess the kinetics of putrescine uptake and metabolism after intraperitoneal or intravenous injection of radiolabeled putrescine (1.2 mmol/kg, specific activity 1 microCi/mmol) into rats fed 36% ethanol diets or isocaloric, nonethanol diets for 6 weeks prior to partial hepatectomy. After putrescine treatment, hepatic putrescine concentrations were greater in ethanol-fed rats than controls. Differences in post-treatment hepatic putrescine levels between ethanol and pair-fed groups could not be explained by differences in the rates of hepatic putrescine uptake or excretion into bile; residual de novo synthesis of putrescine from ornithine or metabolism of hepatic putrescine to its polyamine products, spermidine and spermine. Indeed, supplemental putrescine was not appreciably converted to spermidine or spermine in either ethanol or control rats. Hence, these latter polyamines are unlikely to be responsible for the treatment-associated improvement in DNA synthesis that has been noted in ethanol-fed rats. This suggests that putrescine itself acts to restore hepatic DNA synthesis in ethanol-fed rats.

Effects of vasopressor hormones and modulators of protein kinase C on glutathione efflux from perfused rat liver.

Vasopressor hormones alter efflux of glutathione (GSH) and increase permeability of tight junctions in perfused rat liver. Infusions of 10 nM angiotensin II, 10 microM phenylephrine, and 10 nM vasopressin significantly increased efflux of GSH into perfusate by 32-41% and decreased biliary efflux by 31-57%. Direct modulation of protein kinase C (PKC) activity by 600 nM phorbol 12,13-dibutyrate (PDB), 5 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), 5 microM sphingosine, or 10 nM staurosporine altered the pattern of efflux of GSH but not biliary oxidized glutathione disulfide (GSSG)-GSH ratios. Phorbol dibutyrate mimicked the vasopressor-mediated effects, increasing perfusate efflux by 31% and decreasing biliary efflux by 45%. Inhibitors of PKC caused qualitatively opposite responses, changing perfusate GSH by -37 to 18% and increasing biliary efflux by 22-161%. Whereas vasopressin increased penetration of [14C]sucrose into bile, modulation of PKC activity by PDB and H-7 did not affect the permeability of tight junctions to [14C]sucrose. Although pretreatment with H-7 blocked vasopressin-mediated changes in efflux of GSH, it did not prevent the increase in [14C]sucrose penetrance. We conclude that alterations in sinusoidal and biliary efflux of GSH can occur independent of changes in permeability of hepatocellular tight junctions. These findings suggest a role for protein kinase C in modulating the hepatic efflux of GSH.

Therapeutic ratings and end-of-phase II conferences: initiatives to accelerate the availability of important new drugs.

To facilitate the availability of important new therapeutic agents, the Food and Drug Administration (FDA) in the mid-1970s began assigning therapeutic ratings to investigational new drugs and holding end-of-phase II conferences with drug sponsors. To determine whether these initiatives are associated with faster approvals, we examined new drug application (NDA) review times of new chemical entities (NCEs) approved during the 12-year period 1978 through 1989. Mean NDA review time for 1A drugs (22.5 months) was 22% shorter than that for 1B drugs (28.7 months), which in turn was 25% shorter than that for 1C drugs (38.4 months). For drugs approved during the recent 4-year period 1986 through 1989, however, the gap between 1A and 1C review times has narrowed considerably from 19 to 9 months. When drugs were grouped by FDA reviewing division, 1A drugs had the shortest mean review time in each division except the Cardio-Renal Division; in that division, 1B drugs had the shortest mean review time. Mean NDA review time for drugs that had end-of-phase II conferences (28.6 months) was 15% shorter than that for drugs without such conferences (33.7 months). These results suggest that NCEs that receive 1A or 1B ratings and are the subject of end-of-phase II conferences benefit by having shorter review times.

Glutathione metabolism in normal and cystinotic fibroblasts.

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.

Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions.

Normal leucocyte lysosome-rich granular fractions exhibited counter-transport of cystine, confirming that cystine transport across the lysosomal membrane is carrier-mediated. The trans-activation of cystine transport was temperature-dependent but relatively independent of the external Na+ or K+ concentration in phosphate buffer. Counter-transport, measured as uptake of exogenous [3H]cystine, increased with increasing intralysosomal cystine content up to approx. 3 nmol of half-cystine/unit of hexosaminidase activity. The amount of [3H]cystine entering lysosomes loaded with unlabelled cystine decreased when unlabelled cystine was added to the extralysosomal medium. Lysosomal cystine counter-transport was stereospecific for the L-isomer. Cystathionine, cystamine and cysteamine-cysteine mixed disulphide gave evidence of sharing the lysosomal cystine-transport system, although at lower activity than cystine. Other tested amino acids, including arginine, glutamate and homocystine, were inactive in this system. Nine leucocyte lysosome-rich preparations from eight different cystinotic patients displayed virtually no counter-transport of cystine, conclusively establishing that a carrier-mediated system for cystine transport is dysfunctional in cystinotic lysosomes.

Patterns of amino acid efflux from isolated normal and cystinotic human leucocyte lysosomes.

This study describes the first direct measurements of amino acid efflux from human lysosomes. Isolated leucocyte lysosomes can be loaded with radioactive amino acids by exposure to low concentrations of the corresponding labeled amino acid methyl esters. Efflux of amino acid from the loaded lysosomes can then be determined. Conditions during loading are adjusted for each ester to permit its adequate intralysosomal hydrolysis and subsequent accumulation of the free amino acid. Relative rates of efflux were leucine approximately equal to phenylalanine greater than methionine greater than tryptophan much greater than cystine. Efflux of leucine, tryptophan, or cystine was independent of exogenous cation, ATP, or amino acid concentrations under the conditions tested. Leucine efflux was similar in normal and cystinotic lysosomes, providing strong evidence that isolated cystinotic lysosomes do not manifest a generalized defect in amino acid efflux. In both normal and cystinotic lysosomes, cystine efflux was much slower than efflux of leucine or other amino acids from human or rat liver lysosomes. Significant differences in mean cystine efflux between isolated normal and cystinotic lysosomes were not apparent in the present test system, although the possibility of differences in rats could not be excluded.