Triangulating evidence from longitudinal and Mendelian randomization studies of metabolomic biomarkers for type 2 diabetes.

The number of people affected by Type 2 diabetes mellitus (T2DM) is close to half a billion and is on a sharp rise, representing a major and growing public health burden. Given its mild initial symptoms, T2DM is often diagnosed several years after its onset, leaving half of diabetic individuals undiagnosed. While several classical clinical and genetic biomarkers have been identified, improving early diagnosis by exploring other kinds of omics data remains crucial. In this study, we have combined longitudinal data from two population-based cohorts CoLaus and DESIR (comprising in total 493 incident cases vs. 1360 controls) to identify new or confirm previously implicated metabolomic biomarkers predicting T2DM incidence more than 5 years ahead of clinical diagnosis. Our longitudinal data have shown robust evidence for valine, leucine, carnitine and glutamic acid being predictive of future conversion to T2DM. We confirmed the causality of such association for leucine by 2-sample Mendelian randomisation (MR) based on independent data. Our MR approach further identified new metabolites potentially playing a causal role on T2D, including betaine, lysine and mannose. Interestingly, for valine and leucine a strong reverse causal effect was detected, indicating that the genetic predisposition to T2DM may trigger early changes of these metabolites, which appear well-before any clinical symptoms. In addition, our study revealed a reverse causal effect of metabolites such as glutamic acid and alanine. Collectively, these findings indicate that molecular traits linked to the genetic basis of T2DM may be particularly promising early biomarkers.

Metabolomic biomarkers in urine of rats following long-term low-dose exposure of cadmium and/or chlorpyrifos.

Heavy metals and pesticides can be easily enriched in food chains and accumulated in organisms, thus pose significant threat to human health. However, their combined effects for long-term exposure at low dose has not been thoroughly investigated; especially there was no biofluid biomarker available to noninvasively diagnose the toxicosis of the combined exposure of the two chemicals at their low levels. In this study, we investigated the change of urine metabolites of rats with 90-day exposure to heavy metal cadmium (Cd) and/or organophosphorus pesticide chlorpyrifos (CPF) using gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach. Our results showed that the interaction of Cd and CPF mainly displayed an antagonistic effect. We identified the panels of metabolite biomarkers in urine: benzoic acid and mannose were unique biomarkers for Cd exposure; creatinine and N-phenylacetyl glycine were unique biomarkers for CPF exposure; anthranilic acid, ribitol, and glucose were unique biomarkers for Cd plus CPF exposure. Our results suggest that 90-day exposure to Cd and/or CPF could cause a disturbance in energy and amino acid metabolism. And urine metabolomics analysis can help understand the toxicity of low dose exposure to mixed environmental chemicals.

Enzymatic assay of D-mannose from urine.

AIM: Urinary tract infections (UTIs) are increasingly antibiotic resistant, and alternate or adjunct therapies are urgently needed. Several studies suggest that D-mannose ingestion and a hypothesized increase in urinary D-mannose reduce UTI frequency. Our goal was to develop a reliable assay for urinary D-mannose, which is needed to assess the effects of supplemental D-mannose on urinary D-mannose and UTIs. RESULTS: We developed an enzymatic assay for D-mannose in urine. Hexoses in urine were phosphorylated, sequentially isomerized and oxidized, and the increases in reduced NADPH were measured in a spectrophotometer. Urinary mannose from ten volunteers was well above the detection limit and ranged from 8 to 700 µM. CONCLUSION: A rapid, reliable, and sensitive assay was developed, readily detected urinary D-mannose, and is adaptable to high-throughput analysis. If urinary D-mannose is shown to correlate with susceptibility to UTIs, then the assay could assess susceptibility to UTIs and direct mannose therapy.

Coexisting variants in OSTM1 and MANEAL cause a complex neurodegenerative disorder with NBIA-like brain abnormalities.

Coexistence of different hereditary diseases is a known phenomenon in populations with a high consanguinity rate. The resulting clinical phenotypes are extremely challenging for physicians involved in the care of these patients. Here we describe a 6-year-old boy with co-occurrence of a homozygous splice defect in OSTM1, causing infantile malignant osteopetrosis, and a loss-of-function variant in MANEAL, which has not been associated with human disease so far. The child suffered from severe infantile-onset neurodegeneration that could not be stopped by bone marrow transplantation. Magnetic resonance imaging demonstrated global brain atrophy and showed hypointensities of globus pallidus, corpora mamillaria, and cerebral peduncles, which were comparable to findings in neurodegeneration with brain iron accumulation disorders. LC-MS/MS analysis of urine and cerebrospinal fluid samples revealed a distinct metabolic profile with accumulation of mannose tetrasaccharide molecules, suggestive of an oligosaccharide storage disease. Our results demonstrate that exome sequencing is a very effective tool in dissecting complex neurological diseases. Moreover, we suggest that MANEAL is an interesting candidate gene that should be considered in the context of neurological disorders with brain iron accumulation and/or indications of an oligosaccharide storage disease.

Hyperamylasemia is associated with increased intestinal permeability in patients undergoing diagnostic oral double-balloon enteroscopy.

AIM: To investigate the correlations between serum amylase levels, intestinal permeability (IP), and pancreatic injury and to explore the mechanisms responsible for hyperamylasemia in double-balloon enteroscopy (DBE). METHODS: A prospective study was conducted in 20 patients who underwent DBE from August 1, 2008 to February 28, 2009. Serum amylase was examined 0, 2, 6 and 24 h post-DBE, C-reactive protein and lipase were examined at 24 h, and urine lactulose, mannitol, and trypsinogen-II (TRY-II) levels were measured at 6 h. Lactulose/mannitol ratio indicated IP, and TRY-II indicated pancreatic injuries. Procedure duration and enteroscope insertion length were recorded. RESULTS: Twelve patients underwent oral DBE (M:F, 5:7; mean age 50.42 ± 11.11 years) and 8 underwent anal DBE (M:F, 5:3; mean age 44.75 ± 12.66 years). They all showed significantly increased post-DBE serum amylase. Amylase and lipase levels were higher in the oral DBE group (P < 0.05). Hyperamylasemia was diagnosed in 9 (75.0%) patients undergoing oral DBE. Only patients receiving oral DBE showed increased post-procedure IP, which correlated with increased serum amylase (r = 0.611, P = 0.035) and procedure duration (r = 0.668, P = 0.018). Adverse events included one oral case with pancreatic injury (elevated TRY-II) and two cases of abdominal discomfort in each group. Pancreatitis was not reported. CONCLUSION: Hyperamylasemia correlates with increased IP and clinically undetectable pancreatic injuries. DBE could cause intestinal mucosa damage, which may result in IP elevation and increased amylase absorption, necessitating improvements and standardization of DBE methods.

Effects of rhein on intestinal epithelial tight junction in IgA nephropathy.

AIM: To investigate the effects of rhein on intestinal epithelial tight junction proteins in rats with IgA nephropathy (IgAN). METHODS: Twenty-eight female Sprague-Dawley rats were randomly divided into four groups (7 per group): Control, IgAN, Rhein-treated, and Rhein-prevented. Bovine serum albumin, lipopolysaccharide and CCl4 were used to establish the rat model of IgA nephropathy. The Rhein-treated group was given rhein from week 7 until the rats were sacrificed. The Rhein-prevented group was given rhein from week 1. Animals were sacrificed at the end of week 10. We observed the changes in the intestinal epithelial tight junctions using transmission electron microscopy, and expression of intestinal epithelial tight junction proteins zona occludens protein (ZO)-1 and occludin by immunofluorescence using laser confocal microscopy. Changes in mRNA and protein expression of ZO-1 and occludin were measured by reverse transcriptase polymerase chain reaction and Western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography (HPLC) for assessing the intestinal permeability. RESULTS: In the control group, the tight junctions lied between epithelial cells on the top of the outer side of the cell membrane, and appeared in dense dotted crystal structures, the neighboring cells were binded tightly with no significant gap, and the tight junction protein ZO-1 and occludin were evenly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in the IgAN group, the structure of the tight junction became obscured and the dotted crystal structures had disappeared; the fluorescence of ZO-1 and occludin was uneven and weaker (5.37 ± 1.27 vs 10.03 ± 1.96, P < 0.01; 4.23 ± 0.85 vs 12.35 ± 4.17, P < 0.01); the mRNA expression of ZO-1 and occludin decreased (0.42 ± 0.19 vs 0.92 ± 0.24, P < 0.01; 0.40 ± 0.15 vs 0.97 ± 0.25, P < 0.01); protein expression of ZO-1 and occludin was decreased (0.85 ± 0.12 vs 1.98 ± 0.43, P < 0.01; 0.72 ± 0.15 vs 1.38 ± 0.31, P < 0.01); and the ratio of urinary lactulose/mannitol increased (3.55 ± 0.68 vs 2.72 ± 0.21, P < 0.01). In the Rhein-prevented and Rhein-treated groups, compared with the IgAN group, the intestinal epithelial tight junctions were repaired; fluorescence of ZO-1 and occludin was stronger (11.16 ± 3.52 and 8.81 ± 2.30 vs 5.37 ± 1.27, P < 0.01; 10.97 ± 3.40 and 9.46 ± 2.40 vs 4.23 ± 0.85, P < 0.01); mRNA of ZO-1 and occludin increased (0.81 ± 0.17 and 0.64 ± 0.16 vs 0.42 ± 0.19, P < 0.01; 0.82 ± 0.22 and 0.76 ± 0.31 vs 0.40 ± 0.15, P < 0.01); protein expression of ZO-1 and occludin was increased (2.07 ± 0.41 and 1.57 ± 0.23 vs 0.85 ± 0.12, P < 0.01; 1.34 ± 0.21 and 1.15 ± 0.17 vs 0.72 ± 0.15, P < 0.01); and the ratio of urinary lactulose/mannitol decreased (2.83 ± 0.43 and 2.87 ± 0.18 vs 3.55 ± 0.68, P < 0.01). CONCLUSION: Rhein can enhance the expression of ZO-1 and occludin, repair damaged tight junctions, and protect the intestinal barrier.

Frequency and factors associated with increased small intestinal permeability in patients with portal hypertension.

AIM: Cirrhosis with portal hypertension (PHT) may be associated with increased small intestinal permeability (SIP), predisposing to malnutrition and bacterial translocation causing septicaemia, endotoxaemia and spontaneous bacterial peritonitis. However, data on SIP in extrahepatic portal venous obstruction (EHPVO), in which PHT occurs without hepatic dysfunction, are scanty. Such studies would help to know the effect of PHT on SIP independent of hepatic dysfunction; hence, we undertook this study. METHODS: A total of 96 patients with PHT (cirrhosis 71, EHPVO 25) underwent evaluation of SIP using urinary lactulose/mannitol excretion ratio over 6 hours after oral administration of 15 mL (10 g) lactulose and 5 g mannitol using 1H-NMR spectroscopy by a method described by us previously. RESULTS: Gender of patients with EHPVO and cirrhosis was comparable but patients with EHPVO were younger in age. The causes of cirrhosis were cryptogenic (n = 22), alcohol (n = 20), post-viral (n = 21) and others (n = 8). Twenty-seven (38%) patients with cirrhosis had ascites. Abnormal SIP was detected in 47 (49%) patients (40/71,56% with cirrhosis vs. 7/25, 28% with EHPVO, p = 0.01). Patients with cirrhosis had a higher urinary lactulose/mannitol excretion ratio than those with EHPVO (0.09, range 0-0.87 mmol vs. 0.05, 0-0.19 mmol; p = 0.008). Patients with abnormal SIP had a higher Child score, and more often had cirrhosis than EHPVO, ascites and deranged liver function. On multivariate analysis, presence of cirrhosis, ascites, high serum bilirubin level and prothrombin time were associated with abnormal SIP. CONCLUSIONS: Cirrhosis was associated with abnormal SIP, which was related to liver dysfunction. However, SIP was normal in patients with EHPVO.

Absorption, distribution and excretion of galactosyl-beta-cyclodextrin and mannosyl-beta-cyclodextrin in rats.

Microanalytical methods were developed for measuring galactosyl-beta-cyclodextrin (Gal-betaCD) and mannosyl (Man)-betaCD in biological matrices of the rat by HPLC with pulsed amperometric detection. Then, using these methods, the absorption, distribution and excretion of intravenously and orally administered Gal-betaCD and Man-betaCD were determined in rats, and compared with those of glucosyl (Glc)-betaCD. The pharmacokinetic behavior of Gal-betaCD, Man-betaCD and Glc-betaCD after intravenous administration (50 mg/kg) was very similar. Within 6 h after intravenous administration, unchanged Gal-betaCD and Man-betaCD recovered in urine accounted for about 90% of each dose. After oral administration (500 mg/kg), 0.37% and 0.38% of Gal-betaCD and Man-betaCD, respectively, were excreted in urine. After intravenous and oral administration of Gal-betaCD and Man-betaCD, the decomposition of Gal-betaCD and Man-betaCD to betaCD in the urine, kidney and liver was greater than that of Glc-betaCD. The sum of the molar concentrations of branched CDs and their decomposition product, betaCD, in the liver at 4 h after intravenous administration of Gal-betaCD and Man-betaCD was greater than that of Glc-betaCD. Furthermore, the inclusion complexes of estriol and betamethasone with Gal-betaCD, Man-betaCD and Glc-betaCD were prepared and their absorption was evaluated after oral administration in rats. The plasma concentrations of the drugs after oral administration of drug-Gal-betaCD and drug-Man-betaCD complexes were the same as those after the administration of drug-Glc-betaCD complexes.

Common reabsorption system of 1,5-anhydro-D-glucitol, fructose, and mannose in rat renal tubule.

1,5-Anhydro-D-glucitol (AG) is a major polyol, 99.9% of which is reabsorbed by the kidney. However, such reabsorption is inhibited by competition with glucose excreted in excess, i.e., glucosuria. Under such conditions, AG is excreted into the urine. We administered various types of sugars to rats by continuous intravenous infusion for two hours to evaluate the competition between AG and these sugars for renal reabsorption in vivo. The reabsorption of AG was significantly inhibited by competition with fructose and mannose. The excretion of AG in the 120 min after a load of 3.64 mmol of fructose was 1.99 +/- 0.33 mumol, that after 3.64 mmol of mannose loading was 2.34 +/- 0.43 mumol. These levels were comparable to the AG excretion observed after the administration of the same amount of glucose (3.87 +/- 0.61 mumol). No competition was observed with sucrose, xylose, myoinositol or galactose. The reabsorption of fructose and mannose was significantly inhibited by the presence of AG (P < 0.001) after a mixed load. Results suggest that AG is reabsorbed in the renal tubule by an AG/fructose/mannose-common transport system that is distinct from the major glucose reabsorption system. These findings may help to clarify the specific transport systems for various sugars in the renal tubule, as well as their physiological importance.

[Anti-bacterial defense mechanism of the urinary bladder. Role of mannose in urine].

Bacterial adherence to mucosa is thought to be an initial and important stage to cause urinary tract infection. Among some mechanisms of bacterial adherence, the role of fimbriae and its receptor is worthy of notice. In particular, type 1 fimbriae, for which mannose is assumed as a receptor, is reported as the most common type and called "common fimbriae". Therefore if a certain amount of mannose is present in urine, it will cover the fimbriae of bacteria and competitively block the bacterial adherence to bladder mucosa. As the first step, we tried to detect mannose in urine by high performance liquid chromatography (HPLC). Sugar can be measured by detecting the fluorescence which is produced by a sugar separated by ion exchange, reacting with arginine at high temperature. The results using standard sugar samples should have highly stable retention time and concentration curve with the minimum detectable mannose concentration of 0.02 microgram. We investigated mannose in urine from 186 cases. Since the mannose peak was often masked by near unidentified peaks, the peak of mannose could be detected only in 80 cases and its concentration could be measured only in 24 cases. Mannose concentration in the urine of the 24 cases was between 2.6 and 108.7 micrograms/ml and in most of cases it was lower than 20 micrograms/ml. Secondary, we examined the possibility of a mannose in urine to prevent bacterial adherence to mucosa by the hemagglutination test using guinea pig erythrocytes and type 1 fimbriated E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary proteoglycan degradation product excretion in patients with rheumatoid arthritis and osteoarthritis.

The excretion of sugar components of glycosaminoglycans in the urine was investigated in 19 healthy controls, 27 patients with rheumatoid arthritis, and 24 with osteoarthritis. Both groups of patients excreted significantly more glucosamine, galactosamine, and mannose than the controls. The total uronic acid excretion, also, was higher in the two groups than in the healthy subjects. The possibility of using this method for the long term follow up of rheumatoid arthritis and osteoarthritis and the response to treatment is discussed.

Mannosidosis. New clinical presentation, enzyme studied, and carbohydrate analysis.

Mannosidosis is a rare inborn error of metabolism characterized by deficiency of the lysosomal enzyme alpha-mannosidase and widespread storage of complex carbohydrate, which is enriched in mannose. Two affected unrelated males, aged 6 and 26 years, are reported. Both had a nonprogressive encephalopathy with moderately severe mental retardation. The older patient showed several unique features, including massive gingival hyperplasia associated with histiocytes containing large amounts of a material with the staining characteristics of glycoprotein. The best determinant of mannose storage proved to be the ratio of mannose to other carbohydrates in urinary polysaccharides. The enzyme deficiency in this disease is most convincingly demonstrated at pH values below 4.0. The ability of zinc to activate the mutant enzyme in vitro offers a possible mode of therapy for this disease. Retarded individuals with a Hurler-like appearance and gum hyperplasia of unknown cause should be screened for alpha-mannosidase deficiency.