Rapid screening for lipid storage disorders using biochemical markers. Expert center data and review of the literature.

BACKGROUND: In patients suspected of a lipid storage disorder (sphingolipidoses, lipidoses), confirmation of the diagnosis relies predominantly on the measurement of specific enzymatic activities and genetic studies. New UPLC-MS/MS methods have been developed to measure lysosphingolipids and oxysterols, which, combined with chitotriosidase activity may represent a rapid first tier screening for lipid storage disorders. MATERIAL AND METHODS: A lysosphingolipid panel consisting of lysoglobotriaosylceramide (LysoGb3), lysohexosylceramide (LysoHexCer: both lysoglucosylceramide and lysogalactosylceramide), lysosphingomyelin (LysoSM) and its carboxylated analogue lysosphingomyelin-509 (LysoSM-509) was measured in control subjects and plasma samples of predominantly untreated patients affected with lipid storage disorders (n=74). In addition, the oxysterols cholestane-3ß,5α,6ß-triol and 7-ketocholesterol were measured in a subset of these patients (n=36) as well as chitotriosidase activity (n=43). A systematic review of the literature was performed to assess the usefulness of these biochemical markers. RESULTS: Specific elevations of metabolites, i.e. without overlap between controls and other lipid storage disorders, were found for several lysosomal storage diseases: increased LysoSM levels in acid sphingomyelinase deficiency (Niemann-Pick disease type A/B), LysoGb3 levels in males with classical phenotype Fabry disease and LysoHexCer (i.e. lysoglucosylceramide/lysogalactosylceramide) in Gaucher and Krabbe diseases. While elevated levels of LysoSM-509 and cholestane-3ß,5α,6ß-triol did not discriminate between Niemann Pick disease type C and acid sphingomyelinase deficiency, LysoSM-509/LysoSM ratio was specifically elevated in Niemann-Pick disease type C. In Gaucher disease type I, mild increases in several lysosphingolipids were found including LysoGb3 with levels in the range of non-classical Fabry males and females. Chitotriosidase showed specific elevations in symptomatic Gaucher disease, and was mildly elevated in all other lipid storage disorders. Review of the literature identified 44 publications. Most findings were in line with our cohort. Several moderate elevations of biochemical markers were found across a wide range of other, mainly inherited metabolic, diseases. CONCLUSION: Measurement in plasma of LysoSLs and oxysterols by UPLC-MS/MS in combination with activity of chitotriosidase provides a useful first tier screening of patients suspected of lipid storage disease. The LysoSM-509/LysoSM ratio is a promising parameter in Niemann-Pick disease type C. Further studies in larger groups of untreated patients and controls are needed to improve the specificity of the findings.

Non-polar lipids accumulate during storage of transfusion products and do not contribute to the onset of transfusion-related acute lung injury.

BACKGROUND AND OBJECTIVES: The accumulation of non-polar lipids arachidonic acid, 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE and 15-HETE during storage of transfusion products may play a role in the onset of transfusion-related acute lung injury (TRALI), a syndrome of respiratory distress after transfusion. MATERIALS AND METHODS: We investigated non-polar lipid accumulation in red blood cells (RBCs) stored for 42 days, plasma stored for 7 days at either 4 or 20°C and platelet (PLT) transfusion products stored for 7 days. Furthermore, we investigated whether transfusion of RBCs with increased levels of non-polar lipids induces TRALI in a 'two-hit' human volunteer model. All products were produced following Dutch Blood Bank protocols and are according to European standards. Non-polar lipids were measured with high-performance liquid chromotography followed by mass spectrometry. RESULTS: All non-polar lipids increased in RBCs after 21 days of storage compared to baseline. The non-polar lipid concentration in plasma increased significantly, and the increase was even more pronounced in products stored at 20°C. In platelets, baseline levels of 5-HETE and 15-HETE were higher than in RBCs or plasma. However, the non-polar lipids did not change significantly during storage of PLT products. Infusion of RBCs with increased levels of non-polar lipids did not induce TRALI in LPS-primed human volunteers. CONCLUSION: We conclude that non-polar lipids accumulate in RBC and plasma transfusion products and that accumulation is temperature dependent. Accumulation of non-polar lipids does not appear to explain the onset of TRALI (Dutch Trial Register - NTR4455).

Heparan sulfate derived disaccharides in plasma and total urinary excretion of glycosaminoglycans correlate with disease severity in Sanfilippo disease.

BACKGROUND: Sanfilippo disease (Mucopolysaccharidosis III) is a neurodegenerative lysosomal disorder characterized by accumulation of the glycosaminoglycan heparan sulfate (HS). MPS III has a large phenotypic variability and early assessment of disease severity is difficult. We investigated the correlation between disease severity and the plasma concentration of HS (pHS, defined by the sum of the heparan sulfate derived disaccharides obtained after enzymatic digestion) and urinary total GAGs level (uGAGs, measured by the dimethylene blue test) in a cross-sectional cohort of 44 MPS III patients. METHODS: Disease severity was established on the basis of the age of complete loss of independent walking and of full loss of speech in all patients. Hazard ratios (HR) were obtained with cox-regression analysis. In order to allow prediction of a severe phenotype based on a cut-off value for pHS, patients were divided in two groups (severely affected and less severely affected) based on predictive mutations or on the age of full loss of speech. Receiver operator characteristics (ROC) were obtained for pHS. RESULTS: pHS and uGAGs were independently and linearly associated with an increased risk of speech loss with a HR of 1.8 (95 % CI 1.3-2.7) per 500 ng/ml increase of HS in plasma (p = 0.002), and a HR of 2.7 (95 % CI 1.6-4.4) per 10 mg/mmol creatinine increase of uGAGs (p < 0.001). pHS and uGAGS were less strongly associated with loss of walking. The area under the ROC curve for pHS was 0.85, indicating good discrimination. CONCLUSION: pHS and uGAGs may be useful biomarkers for prediction of severity in MPS III.

Barth syndrome: an X-linked cause of fetal cardiomyopathy and stillbirth.

OBJECTIVE: Barth Syndrome (BTHS) is an X-linked multisystem disorder (OMIM 302060) usually diagnosed in infancy and characterized by cardiac problems [dilated cardiomyopathy (DCM) ± endocardial fibroelastosis (EFE) ± left ventricular non-compaction (LVNC)], proximal myopathy, feeding problems, growth retardation, neutropenia, organic aciduria and variable respiratory chain abnormalities. We wished to determine whether BTHS had a significant impact on fetal and perinatal health in a large cohort of family groups originating from a defined region. METHOD: Case note review on 19 families originating from the UK and known to the Barth Syndrome Service of the Bristol Royal Hospital for Children. RESULTS: Details are presented on six kindreds (32%) with genetically and biochemically proven BTHS that demonstrate a wider phenotype including male fetal loss, stillbirth and severe neonatal illness or death. In these families, 9 males were stillborn and 14 died as neonates or infants but there were no losses of females. BTHS was definitively proven in five males with fetal onset of DCM ± hydrops/EFE/LVNC. CONCLUSION: These findings stress the importance of considering BTHS in the differential diagnosis of unexplained male hydrops, DCM, EFE, LVNC or pregnancy loss, as well as in neonates with hypoglycemia, lactic acidosis and idiopathic mitochondrial disease.

A randomized clinical trial comparing the effect of basal insulin and inhaled mealtime insulin on glucose variability and oxidative stress.

AIM: To assess the effect of three times daily mealtime inhaled insulin therapy compared with once daily basal insulin glargine therapy on 72-h glucose profiles, glucose variability and oxidative stress in type 2 diabetes patients. METHODS: In an inpatient crossover study, 40 subjects with type 2 diabetes were randomized to receive 9 days of inhaled insulin three times daily before meals or 9 days of glargine administered in the morning before breakfast in a randomized order. During the last 72 h in each phase, glucose was measured with continuous glucose monitoring. Activation of oxidative stress was measured by determining the 15(S)-8-iso-PGF(2alpha)-secretion in 24-h urine samples. RESULTS: Inhaled insulin improved overall and postprandial glucose control significantly better than insulin glargine (p < 0.0001). There was a trend towards a greater reduction in glucose variability (8-9%) in the inhaled group [p = 0.1430 and p = 0.3298 for mean amplitude of glycaemic excursions (MAGEs) and mean of daily differences respectively]. Oxidative stress, estimated by determining the urinary isoprostane excretion (15(S)-8-iso-PGF(2alpha)), was equally reduced from baseline by both treatments. No correlation was found between glucose variability and oxidative stress in both groups. CONCLUSIONS: This study showed a mealtime insulin approach to improve glycaemic control more than a basal insulin approach. These findings indicate also that lowering glucose using insulin treatment lowers oxidative stress over time, at least for the study period of 9 days, in type 2 diabetes patients. Contrary to earlier data, we found no correlation between glucose variability (MAGE) and oxidative stress (15(S)-8-iso-PGF(2alpha)) in this study.

The effect of oral administration of ursodeoxycholic acid and high-dose proton pump inhibitors on the histology of Barrett's esophagus.

Bile acids may play a role in the pathogenesis of Barrett's esophagus (BE). Bile composition can be influenced by oral administration of ursodeoxycholic acid (UDCA). We prospectively investigated the effect of proton pump inhibitors (PPI) supplemented with UDCA in vivo in patients with BE. Patients with no or low-grade dysplasia who were clinically asymptomatic on PPI were eligible for the study. In order to exclude the effects of acid reflux, all patients were initially treated with 40 mg esomeprazole (ESO) twice daily for 6 months and continued on this dose till the end of the study (t = 12 months). During a period of 6 months (t = 6 month - t = 12 month) patients were treated with oral UDCA (600 mg twice daily). Patients underwent endoscopy at t = 0 months, t = 6 months and t = 12 months with multiple biopsies of the distal and proximal BE segment, normal squamous and gastric cardia. In addition, pH was measured at t = 0 months and t = 6 months using a BRAVO wireless pH capsule. Bile was sampled at the beginning of the UDCA treatment and 6 months later (t = 6 month and t = 12 month). All biopsies were reviewed for the extent of metaplasia, dysplasia, and acute and chronic inflammation. In addition, proliferation (Ki67), differentiation (villin, cytokeratins 7 and 20) and inflammation (COX-2) were investigated by immunohistochemistry (IHC). Nine patients (mean age 60 years, median BE length 7 cm) were included, of whom six had no dysplasia and three had low-grade dysplasia. pH measurements revealed a normal acid exposure in most patients at t = 0 and t = 6 months. In addition, bile composition analysis demonstrated the efficacy of UDCA. Combining the results of both phases of the study, no significant changes were seen in any of the histological or IHC parameters. Differentiation and proliferation parameters showed no significant changes. In this study, in BE patients who were clinically asymptomatic on PPI, increasing the PPI dose to the maximum for 6 months followed by the addition of UDCA for 6 months did not result in significant histological or IHC changes in their BE.

Effects of acute dietary weight loss on postprandial plasma bile acid responses in obese insulin resistant subjects.

BACKGROUND & AIMS: Bile acids (BA) are pleiotropic hormones affecting glucose and lipid metabolism. The physiochemical properties of different BA species affect their enterohepatic dynamics and their affinity for bile acid receptors. The BA pool composition is altered in patients with type 2 diabetes and obesity. In this study we used a 2-week very-low-calorie diet (VLCD) to investigate the effects of weight loss on BA pool composition and postprandial dynamics. METHODS: We performed mixed meal tests in obese, insulin resistant subjects before and after the VLCD. We measured postprandial plasma levels of glucose, insulin, BA and the BA-induced enterokine fibroblast growth factor 19 (FGF19). RESULTS: The VLCD decreased weight by 4.5 ± 2.3 kg (p < 0.0001) within 14 days. Weight loss increased peak postprandial deoxycholate (DCA) levels (median [IQR]: 0.90 [0.90] vs. 1.25 [1.35] µmol/L; p = 0.045*). Other BA species, glucose, insulin and FGF19 levels and prandial excursions were not significantly affected. The VLCD decreased resting and postprandial energy expenditure by 7 and 11% respectively. CONCLUSIONS: VLCD induced weight loss increased postprandial DCA peak levels and decreased resting energy expenditure in obese insulin resistant subjects.

Polyunsaturated fatty acid status in treated isovaleric acidemia patients.

BACKGROUND/OBJECTIVES: Nutritional deficiencies are frequently observed when treating patients with inborn errors of metabolism due to an unbalanced diet. Thus far, patients with isovaleric acidemia (IVA) who adhere to a restricted protein diet have not been investigated in this respect. We hypothesize that these patients may have a polyunsaturated fatty acid (PUFA) deficiency, leading to potential clinical complications. SUBJECTS/METHODS: We examined the nutritional status by reporting on potential deficiencies in PUFAs in treated IVA patients. A general clinical chemistry work-up as well as gas chromatography flame ionization detector analysis was performed to determine PUFAs in the plasma of 10 IVA patients. RESULTS: The general clinical chemistry tests did not indicate severe hematological abnormalities or nutritional insufficiencies. We identified a significant reduction in plasma PUFA levels, especially in omega-3 (all acids, P<0.001) and omega-6 (in particular 20:3n-6 P<0.0001 and 20:4n-6 P=0.0005) fatty acids. In addition, an elevation in omega-9 fatty acids, with the exception of 20:3n-9 and C22:1n-9, was not suggestive of complete essential fatty acid deficiency but rather indicative of isolated and/or combined omega-3 and omega-6 fatty acid depletion. CONCLUSIONS: This study emphasizes the potential nutritional insufficiencies that may occur because of therapeutic intervention in IVA.

Oral estradiol decreases plasma homocysteine, vitamin B6, and albumin in postmenopausal women but does not change the whole-body homocysteine remethylation and transmethylation flux.

Estrogens, both endogenous and exogenous, lower the fasting levels of the independent risk factor for cardiovascular disease homocysteine. The mechanism behind this observation remains unclear. In a randomized, placebo-controlled, double-blind study, 25 postmenopausal women with a screening homocysteine concentration above 10 micromol/liter were included. We investigated the influence on homocysteine levels of a 3-month treatment with a daily oral dose of 4 mg 17beta-estradiol (ET) or 4 mg ET combined with 10 mg dydrogesterone (EPT); the comparison group received placebo treatment. We performed primed continuous infusions of L-[2H3-methyl-13C]methionine to assess steady-state flux rates of transmethylation, remethylation, and transsulfuration. Homocysteine concentration relationships with S-adenosylmethionine, S-adenosylhomocysteine, creatinine, albumin, vitamins B6 and B12, and folate status were determined as well. The mean change from baseline in homocysteine concentration by both treatments compared with placebo (ET, -13%; EPT, -10%) was accompanied by a decrease in the concentration of vitamin B6 (ET, -25%; EPT, -38%) and albumin (ET, -7%; EPT, -11%). No significant changes in flux rates were observed. In a .multiltivariate analysis, changes in homocysteine concentration were related to changes in albumin concentration. No relation to other variables was observed. We conclude that the ET- and EPT-induced homocysteine changes in this study were not accompanied by a significant change in methionine-homocysteine flux rates and hypothesize that an estrogen-induced lowering of homocysteine levels is primarily part of a change in albumin metabolism.

Establishment of a CSF bank.

A bank of well-characterized CSF has been established by collecting and storing (-70 degrees C) CSF samples remaining after completion of routine clinical studies. Over 1,700 individual patient samples were collected during a 12-month period. A database derived largely from information down-loaded from existing hospital-based systems includes the results of individual CSF laboratory studies, in addition to the patient age, primary diagnoses, and details of any malignancy. CSF control material is used to verify storage conditions. The CSF bank supplies investigators with CSF handled in a standardized manner for more precise investigation of CNS disease.

Nephrotic proteinuria has No net effect on total body protein synthesis: measurements with (13)C valine.

In nephrotic syndrome, significant amounts of plasma proteins, mostly of hepatic origin, are lost in urine. Total hepatic protein synthesis increases, suggesting that other protein pools must be conserved to maintain steady state. This can be accomplished either by decreased amino acid oxidation or decreased protein synthesis in other organs to replace lost liver-derived proteins. To determine the effect of nephrotic syndrome on total-body protein metabolism, we compared whole-body valine use in seven nephrotic patients and five controls using a primed continuous infusion of [1-(13)C]-valine, with additional priming of NaH(13)CO(3). Plasma [(13)C]-valine, (13)C alpha ketoisovaleric acid, and the expired (13)CO(2) enrichments were used to assess whole-body valine flux, valine oxidation, and nonoxidative valine disposal (NOVD). The valine flux into the blood compartment (97.7 +/- 3.0 versus 95.3 +/- 3.3 micromol/kg/h), oxidation of valine (19.4 +/- 1.9 versus 21.2 +/- 2. 8 micromol/kg/h), and NOVD (78.3 +/- 2.5 versus 74.2 +/- 2.7 micromol/kg/h) were not statistically different in patients compared with controls. Valine oxidation correlated positively with urinary urea excretion (r = 0.70; P = 0.01) in all subjects. Compared with control subjects who have similar urinary urea excretion, nephrotic subjects do not compensate for urinary loss of protein by decreased amino acid oxidation or decreased nonoxidative valine disposal. Previous studies have shown that synthesis of several hepatic proteins increases when subjects are fed the same dietary regime, whereas the present study shows that total-body protein synthesis does not increase. This would imply reduced synthesis of nonhepatic protein pools.

Determination of 13C and 15N enrichments of urea in plasma by gas chromatography-combustion isotope ratio mass spectrometry and gas chromatography-mass spectrometry using the 2-methoxypyrimidine derivative.

We describe a GC-MS and GC-c-IRMS method for the determination of labeled urea tracer enrichments in plasma as a result of combined 13C- and 15N(2)-urea infusion experiments in piglets. Urea was converted into 2-methoxypyrimidine, a stable derivative, suited for analyses by both GC-MS and GC-c-IRMS. Using calibration curves for the respective working ranges (13C-urea: 0-1% APE; 15N(2)-urea: 0-7% MPE) enrichments were established in single point measurements; for 15N(2)-urea as values+/-0.15% MPE (95% confidence interval); for 13C-urea as values+/-0.02% APE (95% confidence interval). 15N(1)-urea enrichments were determined by measurement of the same sample with GC-c-IRMS and GC-MS. Subtraction of the 13C specific GC-c-IRMS data from the nondiscriminating GC-MS data for the sum of 13C- and 15N(1)-urea resulted in 15N(1)-urea enrichments+/-0.15% MPE (95% confidence interval). Application of the method in a combined 13C-urea bolus and 15N(2)-urea primed constant infusion experiment in piglet was demonstrated.

In vivo research with stable isotopes in biochemistry, nutrition and clinical medicine: an overview.

Tracers and kinetic modelling provide the opportunity to follow the movement and to quantify the metabolic fates of biological compounds in vivo. For studies in children and for repeated studies in adults, the use of stable isotopically labelled substrates are preferable and safe. Measurement of isotopic enrichment in biological molecules is highly specific and can be extremely precise. This allows the development of models of biological system dynamics in cells and organs that are otherwise inaccessible for sampling. Applications in biochemistry, nutrition and clinical medicine show the potential of stable isotopes in vivo. Methodology is of paramount importance and includes the choice of bolus studies, (primed) continuous infusions, use of multiple tracers and use of isotopomer information and intrinsic labels. There is no limit to the number and kind of compounds that can be traced. Topics include intermediate metabolism (lipids, proteins and carbohydrates) using hydrogen, carbon and nitrogen labels. In clinical medicine, 13C-breath tests are available for detection of Helicobacter pylori ([13C]urea breath test), and for assessment of a variety of gastro-intestinal and hepatic functions (e.g. [13C]octanoate and mixed [13C]triglyceride breath tests for studies of gastric emptying and fat absorption, respectively). Because theoretical, methodological, physiological and practical aspects are intertwined, in vivo research with stable isotopes demands close collaboration between the clinical researcher and the chemist responsible for the mass spectrometry.

Hyperbaric oxygen diving affects exhaled molecular profiles in men.

Exhaled breath contains volatile organic compounds (VOCs) that are associated with respiratory pathophysiology. We hypothesized that hyperbaric oxygen exposure (hyperoxia) generates a distinguishable VOC pattern. This study aimed to test this hypothesis in oxygen-breathing divers. VOCs in exhaled breath were measured in 10 male divers before and 4h after diving to 9msw (190kPa) for 1h. During the dive they breathed 100% oxygen or air in randomized order. VOCs were determined using two-dimensional gas chromatography with time-of-flight mass spectrometry. Compared to air dives, after oxygen dives there was a significant increase in five VOCs (predominately methyl alkanes). Furthermore, a strong, positive correlation was found between increments in 2,4-dimethyl-hexane and those of 4-ethyl-5-methyl-nonane. Although non-submerged hyperoxia studies on VOCs have been performed, the present study is the first to demonstrate changes in exhaled molecular profiles after submerged oxygen diving. The pathophysiological background might be attributed to either a lipid peroxidation-induced pathway, an inflammatory pathway, or to both.

Chenodeoxycholic acid reduces intestinal permeability in newly weaned piglets.

Piglets are highly susceptible to gut health-related problems. Intravenously administered chenodeoxycholic acid (CDCA) affects gut health mediated through glucagon-like peptide 2 (GLP-2). To test whether CDCA is a suitable feed additive for improving gut health, a trial was performed with newly weaned (21 d) piglets offered a diet with or without 60 mg CDCA/kg feed (n = 24/treatment). Upon weaning, piglets were fasted for 16 h and then intragastrically dosed with 20 g test feed in 40 g water. Subsequently, a jugular blood sample was taken on 45, 90, 135, or 180 min for analysis of GLP-2, peptide YY (PYY), and glucose. Afterwards, piglets were offered the experimental diets ad libitum. On days 3.5, 7.5, and 10.5 after weaning, serum responses to an intragastric dose of lactulose and Co-EDTA were tested at 2 h after dosing in 8 piglets per treatment. Immediately thereafter, piglets were euthanized, intestines were harvested, and permeability was measured ex vivo using the everted gut sac technique with 4 kDa fluorescein isothiocyanato (FITC)-dextran as marker at 25, 50, and 75% of the length of the small intestine. Dietary CDCA did not affect (P > 0.05) ADFI, ADG, G:F, blood glucose, and plasma GLP-2 and PYY. Serum cobalt and lactulose at day 10.5 tended to be lower in CDCA pigs compared with control pigs. Serum cobalt and lactulose concentrations were positively correlated (r = 0.67; P < 0.01). In conclusion, CDCA tended to reduce intestinal permeability at 10.5 d after weaning when fed to newly weaned piglets, implying that CDCA deserves further study as a means for improving intestinal health. The positive correlation found between Co-EDTA and lactulose indicates that both marker molecules measure similar change in permeability.

Glucose fluctuations and activation of oxidative stress in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Glucose fluctuations may help predict diabetic complications. We evaluated the relation between glucose variability and oxidative stress in patients with type 1 diabetes. METHODS: Continuous glucose monitors were inserted subcutaneously in 25 patients. During the measurement, patients collected two 24 h urine samples, while 24 healthy controls collected one 24 h urine sample for determination of 15(S)-8-iso-prostaglandin F2alpha(PGF2alpha) using HPLC tandem mass spectrometry. Mean of the daily differences (MODD), mean amplitude of glycaemic excursions (MAGE) and continuous overlapping net glycaemic action calculated with n hour time-intervals (CONGA-n) were calculated as markers for glucose variability and correlation with 15(S)-8-iso-PGF2alpha excretion was calculated. RESULTS: Median [interquartile range (IQR)] urinary 15(S)-8-iso-PGF2alpha was higher in patients than healthy controls: 161 (140-217) pg/mg creatinine vs 118 (101-146) pg/mg creatinine (p = 0.001). Median (IQR) MODD was 3.7 (3.2-5.0) mmol/l, MAGE 7.6 (6.4-9.0) mmol/l and CONGA-1 2.3 (2.1-2.8) mmol/l. Univariate regression did not reveal an association for MODD (r2 = 0.01), MAGE (0.08) or CONGA-1 (0.07) with 15(S)-8-iso-PGF2alpha excretion, nor was an association revealed when corrected for HbA1c, age, sex and smoking. Spearman correlation coefficients (r) between 15(S)-8-iso-PGF2alpha excretion and MODD, MAGE and CONGA-1 were non-significant: -0.112, -0.381 and -0.177. CONCLUSIONS/INTERPRETATION: We report that there is no relationship between glucose variability and urinary 15(S)-8-iso-PGF2alpha. We also confirm that patients with type 1 diabetes have higher levels of urinary 15(S)-8-iso-PGF2alpha than healthy controls, suggesting that in addition to glucose variability, other factors favouring oxidative stress may exist. We did not see a relation between high glucose variability and elevated levels of oxidative stress in patients with type 1 diabetes.

[Treatment of the Achilles tendon rupture by the Ma and Griffith method]. / Leczenie zerwan sciegna Achillesa metoda Ma i Griffitha.

The method has been presented and the results in 13 patients have been given. The results have been compared with those obtained in 8 patients treated by the open method. A more favorable evaluation has been achieved by the patients treated by the closed method.

A study of the positive and negative ion fast atom bombardment mass spectra of alpha-amino acids.

The fast atom bombardment mass spectra of 24 alpha-amino acids have been studied. These include the mass spectra as well as the metastable ion MI and collisional activation CA spectra of [M + H]+ and [M - H]- ions. The extent of the common neutral losses as NH3, H2O and CO2H2 in the positive ion spectra is governed by the nature of the side chain. The relationship between the fragmentation behaviour of the negative ions and the presence of particular functional groups is less obvious. Mixtures of amino acids in glycerol show pronounced surface effects.

Fast atom bombardment tandem mass spectrometry for amino acid sequence determination in tripeptides.

Mass spectral information obtained from the spectra of 26 tripeptides was carefully studied. The data were obtained from their mass, metastable ion and collisional activation spectra. On the basis of the positive ion tandem mass spectra a simple and unambiguous method for the sequence determination of amino acids in tripeptides and Y3" ions is proposed. The use of negative ion spectra for sequence determination in tripeptides is evaluated.