Spin density projection-assisted R2 magnetic resonance imaging of the liver in the management of body iron stores in patients receiving multiple red blood cell transfusions: an audit and retrospective study in South Australia.

AIM: To assess the impact of non-invasive monitoring of liver iron concentration (LIC) on management of body iron stores in patients receiving multiple blood transfusions. METHOD: A retrospective audit was conducted on clinical data from 40 consecutive subjects with haemolytic anaemias or ineffective haematopoiesis who had been monitored non-invasively for LIC over a period of at least 1 year. LIC was measured with spin density projection-assisted proton transverse relaxation rate-magnetic resonance imaging. RESULTS: Nineteen clinical decisions were explicitly documented in the case notes as being based on LIC results. Decisions comprised initiation of chelation therapy, increasing chelator dose, decreasing chelator dose and change of mode of delivery of deferioxamine from subcutaneous to intravenous. The geometrical mean LIC for the cohort dropped significantly (P= 0.008) from 6.8 mg Fe/g dry tissue at initial measurement to 4.8 mg Fe/g dry tissue at final measurement. The proportion of subjects with LIC in the range associated with greatly increased risk of cardiac disease and death (>15 mg Fe/g dry tissue) dropped significantly (P= 0.01) from 14 of 40 subjects at initial measurement to 5 of 40 subjects at final measurement. No significant changes in the geometrical mean of serum ferritin or the proportion of subjects with serum ferritin above 2500 or 1500 µg/L were observed. CONCLUSIONS: The data are consistent with previous observations that introduction of non-invasive monitoring of LIC can contribute to a decreased body iron burden through improved clinical decision making and improved feedback to patients and hence improved adherence to chelation therapy.

Effects of carnitine supplementation on muscle metabolism by the use of magnetic resonance spectroscopy and near-infrared spectroscopy in end-stage renal disease.

BACKGROUND/AIM: A defect in skeletal muscle mitochondrial metabolism develops in patients with chronic renal failure on haemodialysis. Treatment with carnitine, a compound essential for normal mitochondrial function, has been suggested to have significant benefits in such patients, so we carried out a study to see if carnitine acts by improving muscle bioenergetics and function. METHODS: In a phase II randomised double-blind trial, patients with end-stage renal disease received placebo or intravenous L-carnitine (20 mg/kg dry body weight three times weekly after haemodialysis) for 16 weeks (n = 13 in each group). 31P magnetic resonance spectroscopy, 1H magnetic resonance imaging and near-infrared spectroscopy were used to measure muscle bioenergetics and function at baseline and at 16 weeks. RESULTS: There were no significant differences between groups at baseline. Mean plasma carnitine rose 10-fold in the carnitine group but was unchanged in the placebo group. L-carnitine had no statistically significant effect on any of the parameters measured. The rate of proton efflux from muscle, as a measure of tissue perfusion, was low in both groups and was not affected by treatment. CONCLUSIONS: The study failed to show any significant effect of 16 weeks' L-carnitine supplementation on these objective measures of muscle metabolism and function. Slow proton efflux from muscle provides evidence supporting low blood flow and, therefore, decreased oxygen availability, as an underlying mechanism for muscle mitochondrial dysfunction in this disorder.

Cessation of feline calicivirus shedding coincident with resolution of chronic gingivostomatitis in a cat.

Feline calicivirus (FCV) shedding and oral bacterial flora were monitored over a period of 22 months in a case of feline gingivostomatitis (FGS). The cat was treated daily with 50 mg thalidomide capsules by mouth, and 200 mg lactoferrin powder was applied directly to the lesions. Clinical signs began to resolve after 11 months when, in addition to treatment, the diet had been changed to an additive-free cat food supplemented with antioxidant vitamins A, D3 and E. Resolution of clinical signs of FGS coincided with the cessation of FCV shedding, and this is the first report documenting such an association. Which part of the treatment, if any, contributed to the cure requires further investigation.

Antioxidant treatment improves in vivo cardiac and skeletal muscle bioenergetics in patients with Friedreich's ataxia.

Friedreich's ataxia (FA) is the most common form of autosomal recessive spinocerebellar ataxia and is often associated with a cardiomyopathy. The disease is caused by an expanded intronic GAA repeat, which results in deficiency of a mitochondrial protein called frataxin. In the yeast YFH1 knockout model of the disease there is evidence that frataxin deficiency leads to a severe defect of mitochondrial respiration, intramitochondrial iron accumulation, and associated production of oxygen free radicals. Recently, the analysis of FA cardiac and skeletal muscle samples and in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) has confirmed the deficits of respiratory chain complexes in these tissues. The role of oxidative stress in FA is further supported by the accumulation of iron and decreased aconitase activities in cardiac muscle. We used 31P-MRS to evaluate the effect of 6 months of antioxidant treatment (Coenzyme Q10 400 mg/day, vitamin E 2,100 IU/day) on cardiac and calf muscle energy metabolism in 10 FA patients. After only 3 months of treatment, the cardiac phosphocreatine to ATP ratio showed a mean relative increase to 178% (p = 0.03) and the maximum rate of skeletal muscle mitochondrial ATP production increased to 139% (p = 0.01) of their respective baseline values in the FA patients. These improvements, greater in prehypertrophic hearts and in the muscle of patients with longer GAA repeats, were sustained after 6 months of therapy. The neurological and echocardiographic evaluations did not show any consistent benefits of the therapy after 6 months. This study demonstrates partial reversal of a surrogate biochemical marker in FA with antioxidant therapy and supports the evaluation of such therapy as a disease-modifying strategy in this neurodegenerative disorder.

Sympathetic denervation of the upper limb improves forearm exercise performance and skeletal muscle bioenergetics.

BACKGROUND: Sympathetic activation may limit exercise performance by restraining muscle blood flow or by negatively affecting skeletal muscle metabolic behavior. To test this hypothesis, we studied the effect of thoracoscopic sympathetic trunkotomy (TST) on forearm exercise duration, blood flow, and muscle bioenergetics in 13 patients with idiopathic palmar hyperhidrosis. METHODS AND RESULTS: Heart rate and beat-by-beat mean arterial pressure were recorded at rest and during right and left rhythmic handgrip before and 4 to 7 weeks after right TST. Forearm blood flow was measured bilaterally at rest and on the right during exercise. Right forearm muscle phosphocreatine content and intracellular pH were assessed by (31)phosphorus magnetic resonance spectroscopy. After right TST, exercise duration increased from 8.9+/-1.4 to 13.4+/-1.8 minutes (P<0.0001) with the right forearm and from 5.7+/-0.4 to 7.6+/-0.9 minutes (P<0.05) with the left (P<0.05 for the interaction between treatment and side). Right forearm blood flow at rest was 66% higher (P<0.01) after right TST, but this difference decreased as the exercise progressed. After right TST, a significant reduction occurred in muscle acidification and phosphocreatine depletion during ipsilateral forearm exercise. This was associated with a significantly reduced mean arterial pressure response to right handgrip, whereas the pressor response to left handgrip did not change. CONCLUSIONS: Sympathetic denervation of the upper limb significantly improves forearm skeletal muscle bioenergetics and exercise performance in patients with idiopathic palmar hyperhidrosis.

Attempted eradication of salmonellosis from a colony of short-tail grey opossums (Monodelphis domestica).

Prior to re-housing a colony of laboratory short-tail grey opossums, the animals were found to harbour salmonella. This paper describes an unsuccessful attempt to eradicate the infection from the colony by means of antibiotic treatment and hygienic measures. A pilot treatment of five animals which received enrofloxacin 10 mg/kg for 5 days appeared to be successful in that no salmonellae were recovered from faeces or organs sampled after treatment. The process was repeated on the whole colony prior to a change of accommodation but 2 animals were found to be still infected, 5 weeks after cessation of treatment.

A new method for simultaneous immunodetection and morphologic identification of individual sources of pollen allergens.

BACKGROUND: Exposure to outdoor allergens has commonly been estimated by collecting airborne particles with a Hirst-type spore trap and then using morphologic criteria to identify the intact pollen grains and fungal spores that are recognized as allergen sources. Several antibody-based blotting or fixation methods have also been developed that enable the counting of amorphous airborne particles carrying allergen, but none of these methods allow the ready association of the released allergen with the morphologically identifiable particle of origin. A method has been developed that uses pressure-sensitive adhesive tape to sample the airborne particles and then allows the immunoidentification of the specific particles that are the allergen sources. OBJECTIVE: Our purpose was to visualize and immunostain the particles carrying pollen allergen that are collected with a volumetric spore trap. METHODS: A Burkard sampler was used to collect airborne particles onto pressure-sensitive adhesive tapes. The particles were permanently fixed between the tape and a protein-binding membrane when the tape was laminated with the membrane. Allergens that elute from the particles onto the membrane were detected with a range of antibodies. Both the particle and associated immunostained allergen were viewed through the transparent tape for final microscopic identification. RESULTS: Polyclonal and monoclonal antibodies and IgE from allergic patients stained allergens in the periphery of particles collected on the tapes. Individual pollen grains and paucimicronic particles were seen with halos of immunostained allergen surrounding them. When IgE was used, the density of immunostaining in the halo surrounding Lolium perenne pollen grains was found to be proportional to the level of Lolium-specific IgE. The method is highly sensitive and can be used to detect different airborne particles that carry allergen. Both the particle and the immunostaining can be subjected to a range of simple measurement techniques. CONCLUSION: Individual particles carrying allergens and antigens were visualized. These particles included intact pollen grains, paucimicronic particles, and fungal spores.

Subtribe-specific monoclonal antibodies to Lolium perenne.

BACKGROUND AND OBJECTIVES: The ability to measure personal exposure to airborne grass pollen is important in the understanding of allergic diseases. Visual identification is time consuming and it is difficult to distinguish between many grass pollens morphologically. Although grass pollens share common allergenic determinants, we attempted to produce monoclonal antibodies that would distinguish between species, tribes and subfamilies of grasses which would allow immunodetection of pollens. METHODS: Monoclonal antibodies raised against Lolium perenne were screened for specificity against an extended panel of grass pollen extracts using standard ELISA techniques and a novel particle blotting assay using whole pollen grains. RESULTS: Antibodies showing specificity ranging from subfamily to part-tribe specificity were raised. The most specific monoclonal antibodies (numbers 4, 13 and 17) had reactivity to Lolium perenne and Festuca elatior but displayed little cross-reactivity to Phalaris arundinaceae and the rest of the Poeae tribe when tested by ELISA and no detectable cross-reaction when tested with particle blotting. CONCLUSION: Monoclonal antibodies that are functionally specific to only two grasses can be produced and used to discriminate between related grass species.

Reduced cytosolic acidification during exercise suggests defective glycolytic activity in skeletal muscle of patients with Becker muscular dystrophy. An in vivo 31P magnetic resonance spectroscopy study.

Becker muscular dystrophy is an X-linked disorder due to mutations in the dystrophin gene, resulting in reduced size and/or content of dystrophin. The functional role of this subsarcolemma protein and the biochemical mechanisms leading to muscle necrosis in Becker muscular dystrophy are still unknown. In particular, the role of a bioenergetic deficit is still controversial. In this study, we used 31p magnetic resonance spectroscopy (31p-MRS) to investigate skeletal muscle mitochondrial and glycolytic ATP production in vivo in 14 Becker muscular dystrophy patients. Skeletal muscle glycogenolytic ATP production, measured during the first minute of exercise, was similar in patients and controls. On the other hand, during later phases of exercise, skeletal muscle in Becker muscular dystrophy patients was less acidic than in controls, the cytosolic pH at the end of exercise being significantly higher in Becker muscular dystrophy patients. The rate of proton efflux from muscle fibres of Becker muscular dystrophy patients was similar to that of controls, pointing to a deficit in glycolytic lactate production as a cause of higher end-exercise cytosolic pH in patients. The maximum rate of mitochondrial ATP production was similar in muscle of Becker muscular dystrophy patients and controls. The results of this in vivo 31P-MRS study are consistent with reduced glucose availability in dystrophin-deficient muscles.

Normal in vivo skeletal muscle oxidative metabolism in sporadic inclusion body myositis assessed by 31P-magnetic resonance spectroscopy.

Sporadic inclusion body myositis (s-IBM) is a chronic inflammatory myopathy of unknown pathogenesis. The common findings of ragged red fibres, cytochrome c oxidase-negative fibres and multiple mitochondrial DNA deletions in the muscle of patients with s-IBM have suggested that a deficit of energy metabolism may be of pathogenic relevance. To test this hypothesis we used 31P magnetic resonance spectroscopy to assess in vivo skeletal muscle mitochondrial function in the calf muscles of 12 patients with definite s-IBM. Eleven patients showed multiple mitochondrial DNA deletions in skeletal muscle and 67% showed ragged red fibres and/or cytochrome c oxidase-negative fibres. T1-weighted MR images showed increased signal intensity in the calf muscle of all patients except one. The involvement of calf muscle was confirmed by 31P magnetic resonance spectroscopy of resting muscle, which disclosed abnormalities in metabolite ratios in all patients. However, muscle oxidative metabolism assessed during recovery from exercise was normal in patients with s-IBM, as maximum rates of mitochondrial ATP production and post-exercise ADP recovery rates were within the normal range in all cases. We conclude that muscle mitochondrial abnormalities are a secondary process and unlikely to play a significant role in the pathogenesis of s-IBM.

Skeletal muscle metabolism in myotonic dystrophy A 31P magnetic resonance spectroscopy study.

We have used 31P magnetic resonance spectroscopy to investigate skeletal muscle bioenergetics in a total of 31 patients with myotonic dystrophy. Results from resting flexor digitorum superficialis and calf muscle showed a significant elevation in the concentration ratio of inorganic phosphate to ATP and a significant reduction in the phosphorylation potential. In addition, in resting calf muscle the concentration ratio of phosphocreatine to ATP was reduced, and the resting intracellular pH and calculated free cytosolic ADP concentration were elevated. In general, the abnormalities observed were more marked in those patients who were more severely affected as judged by their ability to exercise. During aerobic exercise in both calf muscle and flexor digitorum superficialis, phosphocreatine was depleted more rapidly in patients than in control subjects but the muscle acidified less and ADP concentrations were higher. Calculated ATP turnover was significantly elevated. Analysis of the recovery kinetics for phosphocreatine following exercise provides evidence for a small but significant reduction in mitochondrial function. Analysis of the response of flexor digitorum superficialis to ischaemic exercise provides evidence of a reduction in the relative utilization of glycogen to produce ATP which may account, in part, for the reduced acidification seen in exercising muscle in myotonic dystrophy. There was no definite evidence of an alteration in proton handling capacity in this condition.

Correlative MR imaging and 31P-MR spectroscopy study in sarcoglycan deficient limb girdle muscular dystrophy.

We combined magnetic resonance (MR) imaging and phosphorus magnetic resonance spectroscopy (31P-MRS) to study skeletal muscle in seven patients with limb girdle muscular dystrophy (LGMD) with a variable deficiency of the alpha-, beta-, and gamma-sarcoglycan but normal dystrophin expression on muscle biopsy. T1- and T2-weighted spin-echo axial leg images showed the highest degree of fat replacement in soleus, tibialis anterior and peroneal muscles while gastrocnemius and tibialis posterior were less affected. In LGMD patients as a group, calf muscle phosphorylated compound content did not differ from controls, but the cytosolic pH was increased (P = 0.02). The degree of calf muscle fat replacement correlated inversely with cytosolic pH (r = 0.74) and directly with PCr/ATP (r = 0.74). Muscle oxidative metabolism was normal in LGMD patients. Our findings show that primary deficits of sarcoglycan complex lead to specific morphological and metabolic patterns of skeletal muscle involvement.

Effect of creatine on aerobic and anaerobic metabolism in skeletal muscle in swimmers.

OBJECTIVE: To examine the effect of a relatively low dose of creatine on skeletal muscle metabolism and oxygen supply in a group of training athletes. METHODS: 31P magnetic resonance and near-infrared spectroscopy were used to study calf muscle metabolism in a group of 10 female members of a university swimming team. Studies were performed before and after a six week period of training during which they took either 2 g creatine daily or placebo. Calf muscle metabolism and creatine/choline ratios were studied in resting muscle, during plantar flexion exercise (10-15 min), and during recovery from exercise. RESULTS: There was no effect of creatine on metabolite ratios at rest or on metabolism during exercise and recovery from exercise. Muscle oxygen supply and exercise performance were not improved by creatine if compared to placebo treated subjects. CONCLUSIONS: Oral creatine supplementation at 2 g daily has no effect on muscle creatine concentration, muscle oxygen supply or muscle aerobic or anaerobic metabolism during endurance exercise.

Changes in energy metabolism of calf muscle in patients with intermittent claudication assessed by 31P magnetic resonance spectroscopy: a phase II open study.

Energy status and metabolism in skeletal muscle of nine patients with peripheral arterial disease and suffering from intermittent claudication were evaluated using 31phosphorus magnetic resonance spectroscopy (MRS) before and after treatment for 3 months with propionyl-L-carnitine (PLC; 2 g/day p.o.). Maximum walking distance (MWD) was assessed on a standard treadmill (4 km/h, zero incline). For the group as a whole 31P MRS results did not change significantly with PLC. Although MWD increased by a mean of 36%, this change did not reach significance. However, when these variables were assessed with respect to the change in MWD, there were significant differences between those who increased MWD by > 30% (responders, R; n = 5) and those who did not (nonresponders, NR; n = 4). Compared with pretreatment values, during exercise the decrease in muscle pH in R relative to the decrease in phosphocreatine was less after PLC (p = 0.04). After exercise there was a significant inverse correlation between the changes in recovery half-time (t1/2) for phosphocreatine and in MWD (r = -0.91, p = 0.01). With PLC, Vmax increased in R (p = 0.04), but not in NR. For the patient group as a whole, the changes in Vmax and MWD correlated positively (r = 0.90, p = 0.01). This study helps to identify the changes in muscle metabolism that correlate with changes in exercise performance, and may accompany treatment with PLC.

A relationship between impaired fetal growth and reduced muscle glycolysis revealed by 31P magnetic resonance spectroscopy.

Thinness at birth is associated with insulin resistance and an increased prevalence of non-insulin-dependent diabetes mellitus in adult life. As muscle is an important site of insulin resistance, and because thin babies have reduced muscle mass, thinness at birth may affect muscle structure and function and impair carbohydrate metabolism. We have therefore used 31P magnetic resonance spectroscopy to investigate the bioenergetics of gastrocnemius and flexor digitorum superficialis muscles in 16 normoglycaemic women who had a low ( < or = 23 kg/m3) and 9 women who had a high (> 23 kg/m3) ponderal index at birth. In the flexor digitorum superficialis study anaerobic metabolism was stressed with a constant heavy workload. Low ponderal index subjects fatigued more rapidly (3.3 vs 5.8 min); as phosphocreatine decreased, the accompanying drop in muscle pH was less than in the high ponderal index group. In the first minute of exercise phosphocreatine fell and adenosine diphosphate rose more rapidly (p=0.04 and 0.03, respectively). Gastrocnemius showed a similar trend late in exercise (this exercise was more oxidative, becoming more anaerobic with increasing workload). These changes were not explained by differences in body composition, muscle mass or blood flow. The findings are consistent with a decreased lactic acid and glycolytic adenosine triphosphate production in the low ponderal index group and suggest the possibility that the mechanisms which control substrate utilisation and metabolism in adult life be programmed during prenatal life.

ATP production and mechanical work in exercising skeletal muscle: a theoretical analysis applied to 31P magnetic resonance spectroscopic studies of dialyzed uremic patients.

31P magnetic resonance spectroscopy (31P MRS) can yield much information about bioenergetics in skeletal muscle. During mixed aerobic/glycolytic exercise, changes in phosphocreatine (PCr) concentration and pH may be abnormal because of reduced muscle mass or reduced efficiency (which the authors combine here as "effective muscle mass") or because of reduced oxidative capacity. The authors show how these can be distinguished by calculating the nonoxidative and oxidative costs of mechanical work, and also of work per unit of effective muscle mass (measured using the initial rate of ATP turnover). These quantities are substantially time-independent during incremental exercise, and so can be used to compare exercise studies of differing duration. The authors illustrate this analysis by showing that in dialyzed patients with chronic renal failure, the substantial exercise abnormalities seen by 31P MRS are due mainly to a decrease in effective muscle mass, which outweighs the oxidative defect implied by the abnormal PCr recovery kinetics.

Increased serum proMMP-3 in inflammatory arthritides: a potential indicator of synovial inflammatory monokine activity.

OBJECTIVE: To investigate if the increased concentrations of stromelysin (MMP-3) found in the synovial fluid (SF) of patients with various arthritides reflect the concentrations in the circulation. METHODS: Using a double antibody ELISA, we have measured proMMP-3 concentrations in sera from these patient groups and in others with a heightened acute phase response (APR) as a result of multiple organ failure. RESULTS: The median serum concentration of proMMP-3 was increased by up to ninefold in the inflammatory arthritides, but not in osteoarthritis or in patients with a heightened APR resulting from a non-chronic inflammatory condition. CONCLUSION: In chronic inflammatory diseases such as rheumatoid arthritis, serum proMMP-3 may prove to be a more specific indicator of monokine activity than currently available serum markers.

A 31P magnetic resonance spectroscopy study of mitochondrial function in skeletal muscle of patients with Parkinson's disease.

The activity of complex I of the respiratory chain is decreased in the substantia nigra of patients with Parkinson's disease (PD) but the presence of this defect in skeletal muscle is controversial. Therefore, the mitochondrial function of skeletal muscle in patients with PD was investigated in vivo using 31P magnetic resonance spectroscopy. Results from 7 PD patients, 11 age matched controls and 9 mitochondrial myopathy patients with proven complex I deficiency were obtained from finger flexor muscle at rest, during exercise and in recovery from exercise. In resting muscle, the patients with mitochondrial myopathy showed a low PCr/ATP ratio, a low phosphorylation potential, a high P(i)/PCr ratio and a high calculated free [ADP]. During exercise, stores of high energy phosphate were depleted more rapidly than normal, while in recovery, the concentration of phosphocreatine and free ADP returned to pre-exercise values more slowly than normal. In contrast, the patients with PD were not significantly different from normal for any of these variables, and no abnormality of muscle energetics was detected. Three of the PD patients also had mitochondrial function assessed biochemically in muscle biopsies. No respiratory chain defect was identified in any of these patients by polarography or enzyme analysis when compared with age-matched controls. These results suggest that skeletal muscle is not a suitable tissue for the investigation and identification of the biochemical basis of the nigral complex I deficiency in PD.

No evidence of mitochondrial abnormality in skeletal muscle of patients with iron-deficient anaemia.

OBJECTIVES: Patients with iron deficiency anaemia complain of decreased exercise capacity. We asked whether this is due to defective oxidative ATP synthesis in skeletal muscle as a consequence of reduced blood oxygen content and/or intrinsic mitochondrial abnormalities. DESIGN: We used 31P magnetic resonance spectroscopy to examine skeletal muscle bioenergetics in iron-deficient patients and in age- and sex-matched controls. SETTING: The patients were recruited from the primary care population. SUBJECTS: We studied seven symptomatic female iron-deficient patients (aged 32-70 years) with haemoglobin (Hb) concentration, [Hb], 8.0 g dl-1. Six had menorrhagia, the cause in the seventh patient remained undiagnosed. Results were compared with those of 8 healthy female controls (aged 25-48 years) with mean [Hb] 13.7 g dl-1. RESULTS: The right calf muscle was by studied 31P magnetic resonance spectroscopy in a 1.9 T super-conducting magnet. We measured the intracellular concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), adenosine triphosphate (ATP) and the intracellular pH at rest, during plantar flexion exercise and during recovery from exercise. Exercise duration was reduced in the patients, yet end-exercise PCr/(PCr+Pi) was higher and adenosine diphosphate (ADP) lower than in controls. After exercise, initial PCr recovery was slowed but this was probably because of the lower cytosolic ADP concentration. CONCLUSIONS: Mitochondrial ATP synthesis was not limited by oxygen supply or an intrinsic mitochondrial defect. Therefore, the reduced exercise capacity seen in iron deficiency could be due to central causes and not to skeletal muscle metabolic abnormalities.