Laboratory measurements and astronomical search for cyanomethanimine.

CONTEXT: C-cyanomethanimine (HNCHCN), existing in the two Z and E isomeric forms, is a key prebiotic molecule, but, so far, only the E isomer has been detected toward the massive star-forming region. Sagittarius B2(N) using transitions in the radio wavelength domain. AIMS: With the aim of detecting HNCHCN in Sun-like-star forming regions, the laboratory investigation of its rotational spectrum has been extended to the millimeter-/submillimeter-wave (mm-/submm-) spectral window in which several unbiased spectral surveys have been already carried out. METHODS: High-resolution laboratory measurements of the rotational spectrum of C-cyanomethanimine were carried out in the 100-420 GHz range using a frequency-modulation absorption spectrometer. We then searched for the C-cyanomethanimine spectral features in the mm-wave range using the high-sensitivity and unbiased spectral surveys obtained with the IRAM 30-m antenna in the ASAI context, the earliest stages of star formation from starless to evolved Class I objects being sampled. RESULTS: For both the Z and E isomers, the spectroscopic work has led to an improved and extended knowledge of the spectroscopic parameters, thus providing accurate predictions of the rotational signatures up to ~700 GHz. So far, no C-cyanomethanimine emission has been detected toward the ASAI targets, and upper limits of the column density of ~ 1011-1012 cm-2 could only be derived. Consequently, the C-cyanomethanimine abundances have to be less than a few 10-10 for starless and hot-corinos. A less stringent constraint, ≤ 10-9, is obtained for shocks sites. CONCLUSIONS: The combination of the upper limits of the abundances of C-cyanomethanimine together with accurate laboratory frequencies up to ~ 700 GHz poses the basis for future higher sensitivity searches around Sun-like-star forming regions. For compact (typically less than 1″) and chemically enriched sources such as hot-corinos, the use of interferometers as NOEMA and ALMA in their extended configurations are clearly needed.

Molecular epidemiology of Cryptosporidium spp. and Giardia spp. in mussels (Mytilus californianus) and California sea lions (Zalophus californianus) from Central California.

Cryptosporidium and Giardia are of public health importance, with recognized transmission through recreational waters. Therefore, both can contaminate marine waters and shellfish, with potential to infect marine mammals in nearshore ecosystems. A 2-year study was conducted to evaluate the presence of Cryptosporidium and Giardia in mussels located at two distinct coastal areas in California, namely, (i) land runoff plume sites and (ii) locations near sea lion haul-out sites, as well as in feces of California sea lions (CSL) (Zalophus californianus) by the use of direct fluorescent antibody (DFA) detection methods and PCR with sequence analysis. In this study, 961 individual mussel hemolymph samples, 54 aliquots of pooled mussel tissue, and 303 CSL fecal samples were screened. Giardia duodenalis assemblages B and D were detected in hemolymph from mussels collected near two land runoff plume sites (Santa Rosa Creek and Carmel River), and assemblages C and D were detected in hemolymph from mussels collected near a sea lion haul-out site (White Rock). These results suggest that mussels are being contaminated by protozoa carried in terrestrial runoff and/or shed in the feces of CSL. Furthermore, low numbers of oocysts and cysts morphologically similar to Cryptosporidium and Giardia, respectively, were detected in CSL fecal samples, suggesting that CSL could be a source and a host of protozoan parasites in coastal environments. The results of this study showed that Cryptosporidium and Giardia spp. from the feces of terrestrial animals and CSL can contaminate mussels and coastal environments.

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts.

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.

Radiofrequency-induced thermal inactivation of Toxoplasma gondii oocysts in water.

Toxoplasma gondii, a ubiquitous parasitic protozoan, is emerging as an aquatic biological pollutant. Infections can result from drinking water contaminated with environmentally resistant oocysts. However, recommendations regarding water treatment for oocyst inactivation have not been established. In this study, the physical method of radiofrequency (RF) power was evaluated for its ability to inactivate T. gondii oocysts in water. Oocysts were exposed to various RF energy levels to induce 50, 55, 60, 70 and 80 degrees C temperatures maintained for 1 min. Post-treatment oocyst viability was determined by mouse bioassay with serology, immunohistochemistry and in vitro parasite isolation to confirm T. gondii infections in mice. None of the mice inoculated with oocysts treated with RF-induced temperatures of > or =60 degrees C in an initial experiment became infected; however, there was incomplete oocyst activation in subsequent experiments conducted under similar conditions. These results indicate that T. gondii oocysts may not always be inactivated when exposed to a minimum of 60 degrees C for 1 min. The impact of factors such as water heating time, cooling time and the volume of water treated must be considered when evaluating the efficacy of RF power for oocyst inactivation.

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi).

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters.

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.

Cryptosporidium and Giardia in marine-foraging river otters (Lontra canadensis) from the Puget Sound Georgia Basin ecosystem.

Species of Cryptosporidium and Giardia can infect humans and wildlife and have the potential to be transmitted between these 2 groups; yet, very little is known about these protozoans in marine wildlife. Feces of river otters (Lontra canadensis), a common marine wildlife species in the Puget Sound Georgia Basin, were examined for species of Cryptosporidium and Giardia to determine their role in the epidemiology of these pathogens. Using ZnSO4 flotation and immunomagnetic separation, followed by direct immunofluorescent antibody detection (IMS/DFA), we identified Cryptosporidium sp. oocysts in 9 fecal samples from 6 locations and Giardia sp. cysts in 11 fecal samples from 7 locations. The putative risk factors of proximate human population and degree of anthropogenic shoreline modification were not associated with the detection of Cryptosporidium or Giardia spp. in river otter feces. Amplification of DNA from the IMS/DFA slide scrapings was successful for 1 sample containing > 500 Cryptosporidium sp. oocysts. Sequences from the Cryptosporidium 18S rRNA and the COWP loci were most similar to the ferret Cryptosporidium sp. genotype. River otters could serve as reservoirs for Cryptosporidium and Giardia species in marine ecosystems. More work is needed to better understand the zoonotic potential of the genotypes they carry as well as their implications for river otter health.

Salmonella spp., Vibrio spp., Clostridium perfringens, and Plesiomonas shigelloides in marine and freshwater invertebrates from coastal California ecosystems.

The coastal ecosystems of California are highly utilized by humans and animals, but the ecology of fecal bacteria at the land-sea interface is not well understood. This study evaluated the distribution of potentially pathogenic bacteria in invertebrates from linked marine, estuarine, and freshwater ecosystems in central California. A variety of filter-feeding clams, mussels, worms, and crab tissues were selectively cultured for Salmonella spp., Campylobacter spp., Escherichia coli-O157, Clostridium perfringens, Plesiomonas shigelloides, and Vibrio spp. A longitudinal study assessed environmental risk factors for detecting these bacterial species in sentinel mussel batches. Putative risk factors included mussel collection near higher risk areas for livestock or human sewage exposure, adjacent human population density, season, recent precipitation, water temperature, water type, bivalve type, and freshwater outflow exposure. Bacteria detected in invertebrates included Salmonella spp., C. perfringens, P. shigelloides, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. Overall, 80% of mussel batches were culture positive for at least one of the bacterial species, although the pathogens Campylobacter, E. coli-O157, and Salmonella were not detected. Many of the same bacterial species were also cultured from upstream estuarine and riverine invertebrates. Exposure to human sewage sources, recent precipitation, and water temperature were significant risk factors for bacterial detection in sentinel mussel batches. These findings are consistent with the hypothesis that filter-feeding invertebrates along the coast concentrate fecal bacteria flowing from land to sea and show that the relationships between anthropogenic effects on coastal ecosystems and the environmental niches of fecal bacteria are complex and dynamic.

New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast.

A 3 year study was conducted to evaluate mussels as bioindicators of faecal contamination in coastal ecosystems of California. Haemolymph samples from 4680 mussels (Mytilus spp.) were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Our hypotheses were that mussels collected from sites near livestock runoff or human sewage outflow would be more likely to contain the faecal pathogen Cryptosporidium than mussels collected distant to these sites, and that the prevalence would be greatest during the wet season when runoff into the nearshore marine environment was highest. To test these hypotheses, 156 batches of sentinel mussels were collected quarterly at nearshore marine sites considered at higher risk for exposure to livestock runoff, higher risk for exposure to human sewage, or lower risk for exposure to both faecal sources. Cryptosporidium genotypes detected in Haemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event. Detection of Cryptosporidium spp. was not associated with higher or lower risk status for exposure to livestock faeces or human sewage sources. This study showed that mussels can be used to monitor water quality in California and suggests that humans and animals ingesting faecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.

An unusual genotype of Toxoplasma gondii is common in California sea otters (Enhydra lutris nereis) and is a cause of mortality.

Toxoplasma gondii-associated meningoencephalitis is a significant disease of California sea otters (Enhydra lutris nereis), responsible for 16% of total mortality in fresh, beachcast carcasses. Toxoplasma gondii isolates were obtained from 35 California otters necropsied between 1998 and 2002. Based on multi-locus PCR-restriction fragment length polymorphism and DNA sequencing at conserved genes (18S rDNA, ITS-1) and polymorphic genes (B1, SAG1, SAG3 and GRA6), two distinct genotypes were identified: type II and a novel genotype, here called type x, that possessed distinct alleles at three of the four polymorphic loci sequenced. The majority (60%) of sea otter T. gondii infections were of genotype x, with the remaining 40% being of genotype II. No type I or III genotypes were identified. Epidemiological methods were used to examine the relationship between isolated T. gondii genotype(s) and spatial and demographic risk factors, such as otter stranding location and sex, as well as specific outcomes related to pathogenicity, such as severity of brain inflammation on histopathology and T. gondii-associated mortality. Differences were identified with respect to T. gondii genotype and sea otter sex and stranding location along the California coast. Localised spatial clustering was detected for both type II (centred within Monterey Bay) and x (centred near Morro Bay)-infected otters. The Morro Bay cluster of type x-infected otters overlaps previously reported high-risk areas for sea otter infection and mortality due to T. gondii. Nine of the 12 otters that had T. gondii-associated meningoencephalitis as a primary cause of death were infected with type x parasites.

Isolation of Helicobacter canis from a colony of bengal cats with endemic diarrhea.

On the basis of biochemical, phenotypic, and 16S rRNA analyses, Helicobacter canis was isolated from Bengal cats with and without chronic diarrhea. Because the cats were coinfected with other potential pathogens, including Campylobacter helveticus, and because H. canis was isolated from nondiarrheic cats, the causal role of H. canis in producing the diarrhea could not be proven. Histologically, the colons of the four affected cats were characterized by mild to moderate neutrophilic, plasmacytic, and histocytic infiltrates in the lamina propria. Rare crypt abscesses were also noted for three cats but were a more prominent feature of the colonic lesions noted for the fourth cat. This is the first observation of H. canis in cats and raises the possibility that H. canis, like H. hepaticus and H. bilis in mice, can cause inflammation of the colon, particularly in hosts with immune dysregulation. Further studies are needed to determine the importance of H. canis as a primary enteric pathogen in cats and the role of cats in the possible zoonotic spread of H. canis to humans.

Evaluation of methods to diagnose Clostridium perfringens-associated diarrhea in dogs.

OBJECTIVE: To assess the prevalence of Clostridium perfringens enterotoxin in feces of dogs with and without diarrhea, and to compare the use of microbial cultures from fecal specimens and evaluation of stained fecal smears for endospores with the presence of enterotoxin as tools for diagnosing C perfringens-associated diarrhea. DESIGN: Prospective study. ANIMALS: 144 dogs representing hospitalized dogs with (n = 41) or without (50) diarrhea, and clinically normal dogs treated as outpatients (53). PROCEDURE: Fresh fecal specimens from all dogs were examined as Gram-stained fecal smears to determine numbers of Gram-positive spore-forming rods/100x objective field. Enterotoxin was assayed directly by use of a reverse passive latex agglutination assay. Fecal specimens were plated directly to prereduced egg yolk agar plates and incubated overnight at 37 C in an anaerobic chamber. At 24 hours, up to 3 lecithinase-positive colonies were subcultured to Brucella blood agar to evaluate for double zone hemolysis. Colonies with double zone hemolysis were tested for aerotolerance and Gram-stained. RESULTS: A significant difference was not detected among groups with respect to the presence of C perfringens as determined by culture, the presence of endospores, and the reaction patterns of fecal enterotoxin assays. An association was not found between number of endospores and the presence of fecal enterotoxin. CLINICAL IMPLICATIONS: The presence of C perfringens enterotoxin in feces of dogs, as detected by the latex agglutination assay used in this study, correlates poorly with the number of fecal endospores, regardless of the dog's clinical status.

Sonographic evaluation of liver, spleen, and splanchnic vessels following partial liver resection.

The liver and spleen size and the splanchnic vessel caliber were evaluated by means of real-time ultrasonography in 12 consecutive patients who underwent a partial hepatic resection for benign or malignant lesions. All parameters were evaluated before surgery and 14 days, 28 days, two months, and six months after the partial hepatic resection. The liver size, which was halved after the resection, progressively increased during the follow-up. The splanchnic veins showed, at 14 and 28 days, a significant increase in caliber and a reduced compliance to breathing, which progressively returned to normal levels. The spleen size increased after partial hepatectomy and remained enlarged throughout the study. Ultrasonography was able to detect that partial hepatic resection is followed by a progressive regeneration of the residual parenchyma and by a transient increase in portal pressure, which returns to normal levels when the liver regenerates.

Evaluation of liver volume and liver function following hepatic resection in man.

In a longitudinal study liver volume and liver function were measured in a series of 12 patients undergoing partial liver resection for focal hepatic lesions. Ultrasonography revealed that liver volume, reduced by about 50% by the resection, progressively increased, and 6 months after surgery it returned to nearly normal values. A variable reduction in routine liver function tests was observed, possibly reflecting the influence of the different reserve synthetic capacity of the liver and, in the early postoperative phase, plasma half-life of liver products and blood loss or changes in plasma volume. The galactose elimination capacity was only marginally reduced in the early period (from a pre-surgery value of 2.49 +/- SE 0.21 mmol/min to 2.31 +/- 0.14 after 7 days; p = ns) and reached a nadir at 14 days (1.97 +/- 0.16; p less than 0.001). When expressed per unit of liver volume, the galactose elimination (22 +/- 2 mumol/min per unit before resection) progressively decreased during the regeneration phase from 36 +/- 4 at 14 days to 26 +/- 3 at 6 months (P vs 14-day: less than 0.01). At 6 months both galactose elimination and galactose elimination per unit of liver volume were no longer different from baseline values. Our data show that, following hepatic resection, both liver volume and liver function increase and progressively return to nearly normal values. In agreement with data obtained in animal studies, it appears that the metabolic activity of the remaining parenchyma is increased in the early postoperative phase, and it slows down in the course of regeneration.

Splanchnic vein measurements in patients with liver cirrhosis: a case-control study.

The portal venous system was evaluated by real-time ultrasonography in 100 consecutive cirrhotic patients and 100 pair-matched controls to assess the sensitivity and specificity of ultrasound findings in detecting or excluding cirrhosis. The best discriminant findings were the expiration diameters of the superior mesenteric and the splenic vein and, chiefly, their sum corrected by body surface. In cirrhotics the calibers of the splanchnic veins significantly increase in relation to the extent of esophageal varices, but in individual patients this increase cannot predict the extent of varices, which are the main determinant of the bleeding risk.

Pancreatic beta-cell function in cirrhotic patients with and without overt diabetes. C-peptide response to glucagon and to meal.

To study the role of pancreatic beta-cell function in glucose intolerance and frank diabetes that sometimes develops in cirrhosis, the C-peptide response to a bolus IV injection of 1 mg of glucagon was measured in nine controls and in two groups of patients with cirrhosis. The first group comprised nine subjects with normal or high-normal fasting plasma glucose and no glycosuria; five of them had impaired glucose tolerance. The second group consisted of eight cirrhotics in whom frank diabetes had developed six to 48 months after the diagnosis of cirrhosis. They were characterized by fasting plasma glucose greater than 140 mg/dL and permanent glycosuria. No differences in the degree of liver impairment or portal-systemic shunting were observed between the two groups. Plasma glucose response to glucagon was similarly reduced in cirrhotic subjects. Basal C-peptide was high normal in patients with cirrhosis, and significantly increased in nondiabetic subjects. By contrast peak C-peptide levels and total C-peptide responses to glucagon were low normal in cirrhotics and significantly reduced in patients with cirrhosis and diabetes. In 14 patients the C-peptide response to a standard meal was also measured. It was significantly reduced in patients with cirrhosis and diabetes (six cases), as compared to cirrhotic subjects without diabetes. Peak C-peptide after IV glucagon significantly correlated with peak C-peptide after the meal (r = .927), or total C-peptide response to meal (r = .871). Impaired insulin secretion may add to insulin resistance in patients with liver cirrhosis, leading to the development of frank diabetes, characterized by fasting hyperglycemia and glycosuria.

[Acute experimental hepatic insufficiency in pigs. Validity of a model with biohumoral and electroencephalographic monitoring]. / Insufficienza epatica acuta sperimentale nel suino. Validità di un modello attraverso monitoraggio bioumorale ed elettroencefalografico.

Effectiveness of surgically induced acute hepatic failure in pig and most suitable time to apply artificial support in hepatic coma are evaluated in this work. Five male pigs weighing about 30-35 kg are employed. Latero-lateral porto-caval shunt was performed; the vascular disconnection of liver was obtained by ligature of blood vessels. Ligature was also placed on main biliary way after cholecistectomy. Blood samples were obtained (at 0, 1, 2, 6, 12, 18, 24 hours) to essay serum bilirubin, alkaline phosphatase and GOT-GPT levels as index of cholestasis and necrosis. Porto-caval encephalopathy was evaluated by means of serum ammonium levels, aminoacid pattern and E.E.G. Serum aminoacid pattern was carefully determined; its changes were found similar in man during coma. All pigs died 24-36 hours after surgery with liver ischemic and necrosis. Clinical and laboratory data obtained in experimental conditions were found similar to picture of acute hepatic failure in man, confirming validity of our model.

Plasma amino acid response to protein ingestion in patients with liver cirrhosis.

The metabolic effects of a protein-rich meal were studied for 3 h in 10 controls and in 20 cirrhotic patients. After protein ingestion, blood glucose did not vary significantly. Insulin and glucagon levels rose in controls and, more markedly, in cirrhotics. Aromatic amino acids and tryptophan increased more in cirrhotics as a result of their decreased liver function. Similarly, branched-chain amino acids increased by 153 +/- 14 nmol/ml X min (mean +/- SE) in controls and by 259 +/- 27 nmol/ml X min in cirrhotics (p less than 0.02), in the presence of a markedly increased insulin response. Branched-chain amino acid metabolism mainly occurs in skeletal muscle under insulin control; in cirrhosis, it might be reduced as a consequence of insulin resistance. To support this hypothesis, the effects of the protein meal were compared with those of an oral glucose load in 15 cirrhotic patients. Branched-chain amino acid response to protein ingestion significantly correlated with blood glucose response to oral glucose (r = 0.714), and with insulin resistance during the glucose tolerance test, when assessed by the insulinogenic index (r = 0.628). Similarly, in 8 patients, increased branched-chain amino acid response also correlated with the index of tissue sensitivity to insulin obtained by means of the glucose clamp technique during continuous insulin infusion (r = -0.809). We conclude that liver cirrhosis is characterized by an abnormal branched-chain amino acid response to protein ingestion, which matches the well-known intolerance to oral glucose. Both alterations are possibly due to decreased peripheral insulin activity on substrates.