Plasma norepinephrine in sensory diabetic polyneuropathy.

OBJECTIVE: To examine whether changes in circulating norepinephrine are associated with the sensory disturbances of diabetic polyneuropathy. Experimental studies have indicated that NE can excite sprouts from injured nerves, producing pain. RESEARCH DESIGN AND METHODS: We measured supine and erect plasma NE in 13 normal, nondiabetic control subjects and three groups of diabetic patients: 20 without clinical neuropathy, 20 with chronic painful neuropathy, and 15 with painless neuropathy and foot ulceration. Neuropathy was characterized by symptom and deficit scores, sensory thresholds, electrophysiology, and cardiovascular autonomic function tests. Neuropathic pain was scored by the patients on a linear analogue scale. RESULTS: In painless neuropathy, NE levels were greatly reduced (supine, 1.3 nM; erect, 2.2 nM) compared with control subjects (supine, 2.4 nM; erect, 4.0 nM; P < 0.001) and were combined with grossly abnormal autonomic reflexes. NE also was reduced in the diabetic group without neuropathy (supine, 1.7 nM; erect, 2.7 nM; P < 0.01 vs. control subjects). By contrast, in painful neuropathy NE levels (supine, 2.2 nM; erect, 3.6 nM) were similar to control subjects and significantly higher than in painless neuropathy (P < 0.01). Furthermore, NE correlated with the severity of neuropathic pain (r = 0.46, P = 0.02). To assess whether pain, acting as a stressor, could account for the observed differences in NE, we also measured the stress hormones epinephrine and cortisol. They did not differ among the diabetic groups. CONCLUSIONS: Circulating NE is higher in painful than painless diabetic neuropathy. We suggest that painful neuropathy is associated with a relatively higher number of functioning sympathetic fibers that may contribute to pain.

Biochemical diagnosis of phaeochromocytoma: two instructive case reports.

The biochemical features of two patients with phaeochromocytomas illustrate the inadvisability of depending on a single group of analytes for the diagnosis. The first case presented as a surgical emergency with retroperitoneal haemorrhage. Biochemical diagnosis was difficult since total 24 hour urinary free catecholamine excretion was within normal limits in two out of three samples, and only marginally raised in the third with an atypical preponderance of adrenaline. Plasma catecholamine concentrations were also normal. But urinary excretion of the catecholamine metabolites, metadrenaline and 4-hydroxy-3-methoxy mandelic acid (HMMA), was consistently raised. In contrast, the second patient presenting with headache and labile hypertension showed normal metabolite excretion in the face of grossly increased free noradrenaline excretion and raised plasma noradrenaline concentrations. It is therefore recommend that, as well as urinary free catecholamines, one group of their main metabolites, the 3-methoxy amines (normetadrenaline and metadrenaline) or HMMA, should routinely be measured whenever a phaeochromocytoma is suspected.

Radioenzymatic assay of catecholamines: real and apparent losses of labelled product.

In the radioenzymatic assay of catecholamines, using catechol-O-methyltransferase, the yield of labelled product is frequently less than the expected value. This has been attributed by some workers to losses during the periodate oxidation of the O-methylated derivatives. Using spectrophotometric methods, we have demonstrated that there is no oxidative demethylation of the methoxycatecholamines during periodate oxidation. In a novel technique designed to determine the specific activity of small quantities of tritiated S-adenosylmethionine, high performance liquid chromatography with electrochemical detection was used to measure the amount of adrenaline formed from unlabelled noradrenaline, in the presence of phenylethanolamine-N-methyltransferase and tritiated S-adenosylmethionine. As well as the real losses occurring during solvent extraction and thin layer chromatography (demonstrated spectrophotometrically), apparent 'loss' of radiolabelled product may be due to the assumption that the value for the specific activity of the tritiated S-adenosylmethionine and the determination of the product's radioactivity are both absolutely accurate, with no allowance being made for the normal and expected experimental errors in such measurements.

No significant effects of sodium aurothiomalate on haem metabolism and mixed function oxygenase activity in patients with rheumatoid arthritis.

OBJECTIVE: Animal studies suggest that gold compounds impair haem synthesis and increase haem degradation and, as a result, reduce activity of the hepatic haemoproteins cytochromes P-450. The aim of this study was to investigate whether intramuscular gold exerts similar effects in patients with rheumatoid arthritis (RA). METHODS: Urinary porphyrin and precursor excretion, erythrocyte protoporphyrin, and antipyrine clearance, were measured in 6 patients with RA before and 10 weeks after commencement of intramuscular gold. RESULTS: Parameters of haem metabolism were unaffected by gold. While antipyrine clearance was not statistically changed after gold treatment, in 3 of the patients there was an average decrease in antipyrine clearance of 23%. CONCLUSION: Further studies examining RA patients at different time points are required to investigate further the possibility of reduced hepatic drug metabolising activity during prolonged treatment with gold.

Radioenzymatic assay of catecholamines: reversal of aluminium inhibition of enzymatic O-methylation by desferrioxamine.

Alumina is commonly used for the purification and concentration of catecholamines in biological specimens before analysis. Acid eluates of alumina, found to contain high concentrations of Al3+, interfered with O-methylation but not with N-methylation. The chemistry of the catecholamines, supported by kinetic studies, suggest that complex formation between aluminium and the substrate account for the observed inhibition of O-methylation. The addition of desferrioxamine, a metal-chelating agent, to the reaction mixture reversed this inhibition and, by allowing a preliminary alumina extraction, permits the measurement of low concentrations of catecholamines in biological samples.

Platelet and plasma vasoactive amines in type 1 (insulin-dependent) diabetes mellitus with and without vascular disease.

Platelet and plasma vasoactive amine concentrations were measured in healthy controls and in type 1 (insulin-dependent) diabetic patients with or without vascular disease. Platelet concentrations of serotonin and noradrenaline were similar in all groups and were unrelated to age or gender, or to duration of diabetes, blood pressure, glycaemia or renal function in the diabetic subjects. Plasma concentrations of serotonin in the diabetic groups were comparable (118 +/- 16 (mean +/- SEM) and 127 +/- 21 pmol/mL), and were significantly higher in comparison to the healthy controls (66 +/- 12 pmol/mL, P = 0.002).

Essential fatty acid status in patients on long-term home parenteral nutrition.

BACKGROUND: Patients on total parenteral nutrition are known to be at risk of the development of essential fatty acid deficiency, presenting as a syndrome with scaly skin lesions and characterized by low plasma and erythrocyte linoleic acid concentrations. The essential fatty acid status of patients on long-term home parenteral nutrition who do have access to oral feeds has not been studied. METHODS: With the use of an isocratic high-performance liquid chromatography method, fatty acids were measured in the erythrocytes and plasma of 25 nonfasting patients on long-term home parenteral nutrition and the findings compared with those of 46 hospital outpatients not on nutrition support and five laboratory staff. RESULTS: Statistically significant differences in the two groups were limited to the erythrocytes. Linoleic acid was significantly lower (25.2 vs 40.7 mumol/10(6) red blood cells, p < .0001) and showed a significant correlation with triceps skinfold thickness (r = .52, p = .013). Palmitoleic and oleic acids were higher in patients than controls (10.8 vs 8.4 mumol/10(6) red blood cells, p = .009; 61.2 vs 51.7 mumol/10(6) red blood cells, p = .003). CONCLUSIONS: Despite IV linoleic acid administration, patients on long-term home parenteral nutrition have low erythrocyte stores of this essential fatty acid. This appears to be related to their low body fat stores. We suggest that they may be using much of the infused linoleic acid as an energy source and therefore are at risk of subclinical essential fatty acid deficiency.

Pancreatic islets of malnourished rats: quantitative histologic and electron microscopic findings.

Young rats were maintained for three weeks on a low-protein diet. These animals developed many of the features of human protein-calorie deficiency, including dextrose intolerance and diminished insulin release. Quantitative histologic and ultrastructural studies showed that malnourished rats had (1) a reduced total pancreatic islet volume, and (2) a preponderance of pale granules in the B cells. It is suggested that pale B granules may contain increased amounts of insulin, which accumulate in the cells because of defective insulin release. The mechanism responsible for this has not been elucidated.

Serious hypernatraemia in a hospital population.

Severe hypernatraemia in a hospital population should be an avoidable problem. We have looked at its causes and incidence over one year and have shown that serious hypernatraemia (serum sodium greater than 160 mmol/l) as a manifestation of severe dehydration is associated with a high morbidity and mortality. Failure to maintain adequate fluid intake, intentional or unintentional, was the most frequent cause. Nursing and medical staff must be made more aware of this problem and encouraged to initiate early treatment of dehydration.

Kinetics of enzymatic O-methylation: its value in assays for catecholamines in plasma.

The kinetics of enzymatic O-methylation of catecholamines were studied under conditions like those used in the radioenzymatic assay of plasma catecholamines. Inappropriate Michaelis-Menten kinetics and linear approximations of exponential equations were not used. Mathematical analysis indicated the importance of the ratio of methyl donor (S-adenosylmethionine) to substrate (catecholamine) concentration. If the reaction is incomplete, only a large ratio will allow linear approximations between product formed and initial catecholamine concentration. The use of high-concentration internal standards to correct for plasma interference may give erroneous results by reducing this ratio. Accuracy will be improved by ensuring (a) that S-adenosylmethionine is always greatly in excess of catecholamine, (b) that concentrations of added standards are of the same order as for endogenous catecholamine, and (c) that a high activity of enzyme is used, to allow the reaction to reach completion even in the presence of some inhibition.

Fatty acids in erythrocytes measured by isocratic HPLC.

We developed an isocratic reversed-phase HPLC method to measure arachidonic, palmitoleic, linoleic, eicosatrienoic, oleic, palmitic, and stearic acids from hydrolyzed erythrocytes. Washed erythrocytes were heated in methanol:HCl and the fatty acids extracted into hexane:amyl alcohol. After derivatization with 4-bromomethyl-7-methoxycoumarin, samples diluted in mobile phase (acetonitrile:water, 85:15 by vol) were injected onto a 250 x 4.6 mm C18 column, and the eluted fatty acids were detected fluorometrically. For all analytes, the mean within-batch CV was 8.2% (5.5-10.8%), the mean limit of detection was 7.0 mumol/L, a linear response was maintained up to 400 mumol/L, and results agreed well with those by gas chromatography. The addition of antioxidant (butylated hydroxytoluene) was essential for sample stability. We discuss hydrolysis and extraction times, derivatization temperature, critical steps in chromatography, and concentration units.

Ascorbic acid as an antioxidant in measurements of catecholamines in plasma.

Sodium metabisulfite, commonly used to prevent the oxidation of catecholamines during extraction from plasma onto alkaline alumina, does not prevent their subsequent degradation in acetic acid eluates. However, ascorbic acid, a potent antioxidant, is extracted with the catecholamines onto the alumina and prevents such destruction. However, ascorbic acid may interfere with the electrochemical measurement of catecholamines, unless sequential oxidation and reduction are used. Other methods of minimizing catecholamine oxidation in acetic acid eluates include refrigerating at 4 degrees C and capping the sample vials to exclude atmospheric oxygen.

Glucose tolerance and insulin release in malnourished rats.

1. Young Wistar rats were used as an experimental model to determine the effects of protein-energy malnutrition on glucose tolerance and insulin release. 2. Malnourished rats presented some of the features commonly found in human protein-energy malnutrition, such as failure to gain weight, hypoalbuminaemia, fatty infiltration of the liver and intolerance of oral and intravenous glucose loads. 3. The rate of disappearance of glucose from the gut lumen was greater in the malnourished rats but there was no significant difference in portal blood glucose concentration between normal and malnourished rats 5 and 10 min after an oral glucose load. 4. Insulin resistance was not thought to be the cause of the glucose intolerance in the malnourished animals since these rats had a low fasting plasma insulin concentration with a normal fasting blood glucose concentration and no impairment in their hypoglycaemic response to exogenous insulin administration. Furthermore, fasting malnourished rats were unable to correct the insulin-induced hypoglycaemia despite high concentrations of hepatic glycogen. 5. Malnourished rats had lower peak plasma insulin concentrations than normal control animals after provocation with oral and intravenous glucose, intravenous tolbutamide and intravenous glucose plus aminophyllin. This was not due to a reduction in the insulin content of the pancreas or potassium deficiency. Healthy weanling rats, like the older malnourished rats, had a diminished insulin response to intravenous glucose and intravenous tolbutamide. However, their insulin response to stimulation with intravenous glucose plus aminophyllin far exceeded that of the malnourished rats. Thus the impairment of insulin release demonstrated in the malnourished rats cannot be ascribed to a 'functional immaturity' of the pancreas.

Freedom from drug interference in new immunoassays for urinary catecholamines and metanephrines.

BACKGROUND: Determination of urinary free catecholamine and total (i. e., free plus conjugated) metanephrine excretion is considered the most clinically sensitive biochemical test for pheochromocytoma. In this study, we evaluated new immunoassay methods for the measurement of these analytes for potential drug-based interference. METHODS: Urine samples collected from patients on a variety of medications were grouped by specific drug type. The significance of any difference in the free catecholamine or total metanephrine concentrations in the different groups was assessed by one-way ANOVA. A group of patients receiving no medication was included as a control (no analytical interference). Additionally, analytical accuracy, detection limit, and precision were determined. RESULTS: No significant differences were found in the concentrations of free catecholamines or total metanephrines in urine from patients taking the medications investigated and the control group: P = 0.649 (fE), 0.221 (fNE), 0.149 (tM), and 0.170 (tNM). For free catecholamines, intraassay CVs were 4.6-18%; interassay CVs were 10-25%. For total metanephrines, intraassay CVs were 9.6-27%; interassay CVs were 5. 8-22%. Detection limits were 0.009 and 0.027 micromol/L for fE and fNE and 0.119 and 0.346 micromol/L for tM and tNM, respectively. CONCLUSIONS: None of the drugs examined in this study interfered in the measurement of free catecholamines or total metanephrines by these immunoassays. The technique is easier to use, requires less equipment, and is more accessible than HPLC. In combination, these assays are suitable as initial screening tests for pheochromocytoma.

Suggested guidelines for the use of emergency tests in clinical biochemistry.

We have discussed the need for guidelines as a means of reducing out-of-hours investigations in clinical biochemistry, and suggest protocols that can be modified to suit local needs.