An integrated portable system for single chip simultaneous measurement of multiple disease associated metabolites.

Metabolites, the small molecules that underpin life, can act as indicators of the physiological state of the body when their abundance varies, offering routes to diagnosis of many diseases. The ability to assay for multiple metabolites simultaneously will underpin a new generation of precision diagnostic tools. Here, we report the development of a handheld device based on complementary metal oxide semiconductor (CMOS) technology with multiple isolated micro-well reaction zones and integrated optical sensing allowing simultaneous enzyme-based assays of multiple metabolites (choline, xanthine, sarcosine and cholesterol) associated with multiple diseases. These metabolites were measured in clinically relevant concentration range with minimum concentrations measured: 25 µM for choline, 100 µM for xanthine, 1.25 µM for sarcosine and 50 µM for cholesterol. Linking the device to an Android-based user interface allows for quantification of metabolites in serum and urine within 2 min of applying samples to the device. The quantitative performance of the device was validated by comparison to accredited tests for cholesterol and glucose.

Fenofibrate causes elevation of betaine excretion but not excretion of other osmolytes by healthy adults.

BACKGROUND: Cross-sectional data suggest that bezafibrate increases betaine excretion in dyslipidemic patients. OBJECTIVE: We aimed to demonstrate that fenofibrate induces increased betaine excretion in normal subjects and explore whether other 1-carbon metabolites and osmolytes are similarly affected. METHODS: Urine was collected from 26 healthy adults before and after treatment with fenofibrate (145 mg/day for 6 weeks). Excretions of betaine, N,N-dimethylglycine, free choline, myo-inositol, taurine, trimethylamine-N-oxide, carnitine, and acetylcarnitine were measured by liquid chromatography with mass spectrometric detection. RESULTS: Fenofibrate increased the median betaine excretion from 7.5 to 25.8 mmol/mole creatinine (median increase 3-fold), P < .001. The median increase in N,N-dimethylglycine excretion was 2-fold (P < .001). Median choline excretion increased 12% (significant, P = .029). Participants with higher initial excretions tended to have larger increases (P < .001 in all 3 cases). Fenofibrate did not significantly change the median excretions of myo-inositol, taurine, trimethylamine-N-oxide, and carnitine. The excretion of acetylcarnitine decreased 4-fold on treatment, with no correlation between the baseline and after-treatment excretions. Changes in all urine components tested, except trimethylamine-N-oxide, positively correlated with changes in betaine excretion even when the median excretions before and after were not significantly different. CONCLUSIONS: Fibrates increase betaine, and to a lesser extent N,N-dimethylglycine and choline, excretion. Other osmolytes are not elevated. Because the increase in betaine excretion depends on the baseline excretion, large increases in excretion in the metabolic syndrome and diabetes (where baseline excretions are high) could be expected. Replacement with betaine supplements may be considered.

Extreme urinary betaine losses in type 2 diabetes combined with bezafibrate treatment are associated with losses of dimethylglycine and choline but not with increased losses of other osmolytes.

PURPOSE: Betaine deficiency is a probable cardiovascular risk factor and a cause of elevated homocysteine. Urinary betaine excretion is increased by fibrate treatment, and is also often elevated in diabetes. Does fibrate further increase betaine excretion in diabetes, and does it affect the plasma concentrations and excretions of related metabolites and of other osmolytes? METHODS: Samples from a previous study of type 2 diabetes were selected if participants were taking bezafibrate (n = 32). These samples were compared with participants matched for age and gender and not on a fibrate (comparator group, n = 64). Betaine, related metabolites, and osmolytes were measured in plasma and urine samples from these 96 participants. RESULTS: Median urinary betaine excretion in those on bezafibrate was 5-fold higher than in the comparator group (p < 0.001), itself 3.5-fold higher than the median reported for healthy populations. In the bezafibrate group, median dimethylglycine excretion was higher (9-fold, p < 0.001). Excretions of choline, and of the osmolytes myo-inositol, taurine and glycerophosphorylcholine, were not significantly different between groups. Some participants excreted more betaine than usual dietary intakes. Several betaine fractional clearances were >100 %. Betaine excretion correlated with excretions of the osmolytes myo-inositol and glycerophosphorylcholine, and also with the excretion of choline and N,N-dimethylglycine, but it was inconclusive whether these relationships were affected by bezafibrate therapy. CONCLUSIONS: Increased urinary betaine excretions in type 2 diabetes are further increased by fibrate treatment, sometimes to more than their dietary intake. Concurrent betaine supplementation may be beneficial.

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 induce different age-related metabolic profiles revealed by 1H-NMR spectroscopy in urine and feces of mice.

Age-related dysbioses of intestinal microbiota and decline in the overall metabolic homeostasis are frequently found in the elderly. Probiotic supplementation may represent a way to prevent or reduce the senescence-associated metabolic disorders. The present study evaluated the metabolic impact of Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 supplementation in relation to age by analyzing urine and feces metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy and multivariate analysis. Adult (3 mo old) and aged (16 mo old) mice received an oral supplementation of the 2 probiotics (1 × 10(9) colony-forming units/d each) or phosphate buffered saline (control) daily for 30 d. Urine and feces were collected for 48 h before the end of the study. Partial least squares-discriminant analysis showed that the urinary discriminant metabolites for the probiotic treatment included higher dimethylglycine in adult and aged mice, lower sarcosine and nicotinate in adult mice, higher N-methylnicotinamide in adult mice and lower N-methylnicotinamide in aged mice compared with their controls. These results indicate a probiotic-induced modulation of homocysteine and NAD metabolism pathways, which have important implications because these pathways are involved in essential cellular processes that can be altered in senescence. The probiotic supplementation also modified the fecal metabolic profiles, inducing in both adult and aged mice higher 4-hydroxyphenylacetate and lower xylose in treated mice compared with their control mice, whereas valerate was greater in treated adult mice and lower in treated aged mice compared with their controls. The ANOVA simultaneous component analysis on urinary and fecal metabolic profiling showed an age × treatment interaction (P < 0.05), confirming the age-related modulation of the metabolic response to probiotic supplementation. The results suggest that L. acidophilus and B. lactis may prevent or reduce age-related metabolic dysfunction.

[Urine metabonomic study of intervention effects of Morinda officinalis how. on 'kidney-yang deficiency syndrome'].

To investigate the intervention effects of Morinda officinalis How. on 'Kidney-yang deficiency syndrome' induced by hydrocortisone in rats, the metabolic profiles of rat urine were characterized using proton nuclear magnetic resonance and principal component analysis (PCA) was applied to study the trajectory of urinary metabolic phenotype of rats with 'Kidney-yang deficiency syndrome' under administration of M. officinalis at different time points. Meanwhile, the intervention effects of M. officinalis on urinary metabolic potential biomarkers associated with 'Kidney-yang deficiency syndrome' were also discussed. The experimental results showed that in accordance to the increased time of administration, an obvious tendency was observed that clustering of the treatment group moved gradually closed to that of the control group. Eight potential biomarkers including citrate, succinate, alpha-ketoglutarate, lactate, betaine, sarcosine, alanine and taurine were definitely up- or down-regulated. In conclusion, the effectiveness of M. oficinalis on 'Kidney-yang deficiency syndrome' is proved using the established metabonomic method and the regulated metabolic pathways involve energy metabolism, transmethylation and transportation of amine. Meanwhile, the administration of M. officinalis can alleviate the kidney impairment induced by 'Kidney-yang deficiency syndrome'.

Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine.

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5 × 10(-7) M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory.

MTHFR C677T genotype influences the isotopic enrichment of one-carbon metabolites in folate-compromised men consuming d9-choline.

BACKGROUND: Homozygosity for the variant 677T allele in the methylenetetrahydrofolate reductase (MTHFR) gene increases the requirement for folate and may alter the metabolic use of choline. The choline adequate intake is 550 mg/d for men, although the metabolic consequences of consuming extra choline are unclear. OBJECTIVE: Deuterium-labeled choline (d9-choline) as tracer was used to determine the differential effects of the MTHFR C677T genotype and the effect of various choline intakes on the isotopic enrichment of choline derivatives in folate-compromised men. DESIGN: Mexican American men with the MTHFR 677CC or 677TT genotype consumed a diet providing 300 mg choline/d plus supplemental choline chloride for total choline intakes of 550 (n = 11; 4 with 677CC and 7 with 677TT) or 1100 (n = 12; 4 with 677CC and 8 with 677TT) mg/d for 12 wk. During the last 3 wk, 15% of the total choline intake was provided as d9-choline. RESULTS: Low but measurable enrichments of the choline metabolites were achieved, including that of d3-phosphatidylcholine (d3-PtdCho)--a metabolite produced in the de novo pathway via choline-derived methyl groups. Men with the MTHFR 677TT genotype had a higher urinary enrichment ratio of betaine to choline (P = 0.041), a higher urinary enrichment of sarcosine (P = 0.041), and a greater plasma enrichment ratio of d9-betaine to d9-PtdCho with the 1100 mg choline/d intake (P = 0.033). CONCLUSION: These data show for the first time in humans that choline itself is a source of methyl groups for de novo PtdCho biosynthesis and indicate that the MTHFR 677TT genotype favors the use of choline as a methyl donor.

Dietary and supplementary betaine: effects on betaine and homocysteine concentrations in males.

BACKGROUND AND AIMS: Betaine is an osmolyte that when catabolised decreases plasma total homocysteine. A betaine-rich meal has acute effects similar to a supplement, but the effects of a longer-term increase in dietary betaine intake need clarification. We compared the effects of two weeks of dietary and supplementary betaine on plasma betaine and homocysteine concentrations both fasting and after a methionine load. METHODS AND RESULTS: In a randomized crossover study, 8 healthy males (22-36 y) consumed either a betaine-rich diet ( approximately 800 mg/day) or a betaine supplement (0.5 g twice daily) for 14 days. Fasting blood samples were collected on day -5, -1 (pre-treatment) 0, 2, 6, 9, 13 (treatment), 14 and 18 (post-treatment). Post-methionine load blood samples were collected on day -5, 0, 6 and 13, while 24h urine samples were collected on day -5, 0, 6, 13 and 14. Plasma betaine, dimethylglycine, homocysteine and urine betaine, dimethylglycine and creatinine concentrations were measured. Plasma betaine concentrations significantly increased for both treatments compared to pre-treatment values (P<0.001). Fasting homocysteine levels were minimally affected. Both treatments reduced post-methionine load homocysteine and this effect tended to be greater following a betaine-rich diet (P=0.108). Small increases in urinary betaine excretion were observed following both treatments ( approximately 1.5% of supplement; approximately 1.3% of dietary betaine). Most was attributable to increased excretion of betaine as dimethylglycine. CONCLUSIONS: Supplemental or dietary betaine similarly increase circulating betaine concentrations and attenuate the post-methionine load rise in homocysteine concentrations.

Plasma and urine betaine and dimethylglycine variation in healthy young male subjects.

OBJECTIVES: We aimed to compare the individuality (within subject consistency) of plasma and urine betaine and N,N-dimethylglycine. DESIGN AND METHODS: In two separate groups of 8 males (ages 19 to 40), plasma (10) and urine (6) samples were collected either over a single day or over an 8 week period. The individuality of the betaine and N,N-dimethylglycine plasma concentrations and excretions were estimated by one-way repeated measures analysis of variance. The reliability coefficients and indices of individuality were calculated. The between-subject variation in the study population was compared with that in a normal population (n=192 for plasma, 205 for urine). RESULTS: Plasma betaine concentrations were significantly different between subjects over 24 h and 8 weeks (p<0.00001). Plasma dimethylglycine concentrations were different over 24 h. Urine betaine and dimethylglycine excretions were different in both (p<0.0001). Betaine was more individual than dimethylglycine in both plasma and urine. Compared with a normal healthy population, the between-subject variation in plasma betaine was less (p<0.001) in the study group, but similar for dimethylglycine and for urine betaine. CONCLUSIONS: Plasma betaine and urinary betaine excretions are more individual than dimethylglycine. Plasma and urine betaine are highly individual in the general population.

Dietary and supplementary betaine: acute effects on plasma betaine and homocysteine concentrations under standard and postmethionine load conditions in healthy male subjects.

BACKGROUND: Betaine comes from the diet and from choline, and it is associated with vascular disease in some patient groups. Betaine supplementation lowers plasma total homocysteine. OBJECTIVE: We compared the acute effects of dietary and supplementary betaine and choline on plasma betaine and homocysteine under standard conditions and after a methionine load. DESIGN: In a randomized crossover study, 8 healthy men (19-40 y) consumed a betaine supplement (approximately 500 mg), high-betaine meal (approximately 517 mg), choline supplement (500 mg), high-choline meal (approximately 564 mg), high-betaine and -choline meal (approximately 517 mg betaine, approximately 622 mg choline), or a low-betaine and -choline control meal under standard conditions or postmethionine load. Plasma betaine, dimethylglycine, and homocysteine concentrations were measured hourly for 8 h and at 24 h after treatment. RESULTS: Dietary and supplementary betaine raised plasma betaine concentrations relative to control (P < 0.001) under standard conditions. This was not associated with raised plasma dimethylglycine concentration, and no significant betaine appeared in the urine. A small increase in dimethylglycine excretion was observed when either betaine or choline was supplied (P = 0.011 and < 0.001). Small decreases in plasma homocysteine 6 h after ingestion under standard conditions (P < or = 0.05) were detected after a high-betaine meal and after a high-betaine and high-choline meal. Dietary betaine and choline and betaine supplementation attenuated the increase in plasma homocysteine at both 4 and 6 h after a methionine load (P < or = 0.001). CONCLUSIONS: Dietary betaine and supplementary betaine acutely increase plasma betaine, and they and choline attenuate the postmethionine load rise in homocysteine concentrations.

The importance of recognizing secondary carnitine deficiency in organic acidaemias: case report in glutaric acidaemia type II.

Secondary carnitine deficiency in a patient with glutaric acidaemia type II, due to deficient ETF-dehydrogenase activity, is described. The patient responded clinically to a pharmacological dose of riboflavin and a restricted protein diet. In the second year of her life she developed more frequent and severe exacerbations during intercurrent infections from which she did not fully recover. Hypotonia and marked ataxia persisted. Plasma carnitine was entirely complexed as acylcarnitine with no free carnitine detected. Retrospective evaluation of several frozen urine specimens obtained since the age of 10 months revealed undetectable free carnitine with elevated acylcarnitine levels. Marked clinical improvement was observed following L-carnitine supplementation. The hypotonia and ataxia disappeared. The frequency and the severity of the exacerbations were noticeably decreased. The role of L-carnitine in preventing the accumulation of acyl-CoA compounds in inborn errors of organic acid metabolism is further emphasized by this patient. The necessity to evaluate free carnitine, acylcarnitine and acyl/free ratio in the assessment, follow-up and management of patients with inborn errors of organic acid metabolism is discussed.