The relationship between chronic glycaemic control and oxidative stress in type 2 diabetes mellitus.

This study compares the lipid peroxidation marker urinary thiobarbituric acid reactive substances (TBARS) and antioxidants including plasma alpha-tocopherol (vitamin E), plasma (P-GSH-Px) and erythrocyte glutathione peroxidase (E-GSH-Px) activities, and plasma selenium levels in two groups of type 2 diabetic subjects (both n=20) with a disease duration of < or =2 (GP1) and 4-6 years (GP2), and non-diabetic age and gender-matched control subjects (CG, n=20). The mean (standard deviation [SD]) age of the groups was similar at 41(10) years. Fasting blood and midstream urine samples were obtained from diabetic and non-diabetic subjects attending the diabetic clinic and HbAlc, fructosamine, urine TBARS, total antioxidant (TAS) levels, P-GSH-Px, E-GSHPx and plasma selenium and vitamin E concentrations were measured. HbA1c (%) and fructosamine levels in the GP1 and GP2 diabetic subjects and the controls were 5.75 (0.67), 11.43 (2.01) and 4.33 (0.47), and 3.09 (0.57), 6.09 (1.15) and 1.67 (0.31), respectively (GP1 vs. GP2, GP1 vs. GC and GP2 vs. CG, all P < 0.001). Elevated urinary TBARS (micromol/mmol urinary creatinine) in the GP1, GP2 and GC groups were 2.47 (0.37), 3.73 (0.93) and 1.18 (0.24), respectively (GP1 vs. GP2, GP1 vs. GC and GP2 vs. CG, all P < 0.001). A significant correlation between HbA1c and TBARS was also noted (r2 = 0.894, P < 0.001) but only in the GP2 subjects. TAS levels were only decreased in the GP2 group compared to control values (0.59 [0.18] vs. 1.74 [0.21], P < 0.001). Plasma vitamin E concentrations (micromol/L) of 34.11 (3.31), 9.57 (2.20) and 39.01 (2.91) were observed in the GP1, GP2 and GC groups, respectively (GP1 vs. CG, P < 0.05 and GP1 vs. GP2 and GP vs. CG, both P < 0.001). E-GSH-Px (U/g Hb) and P-GSH-Px (U/L) activities in GP1, GP2 and CG groups were also decreased at 57.04 (4.31), 24.0 (8.94) and 67.6 (4.29), and 6.16 (1.56), 2.67 (0.47) and 8.72 (0.31), respectively (E-GSH-Px: CG vs. GP1, P < 0.01, CG vs. GP2 and GP1 vs. GP2, both P < 0.001; P-GSH-Px: CG vs. GP1, CG vs. GP2 and GP1 vs. GP2, all P < 0.001). Plasma selenium levels (miromol/L) were only significantly decreased in GP2 compared to both GP1 and CG values (0.49 [0.29] vs. 1.67 [0.80] vs. 1.79 [0.26], both P < 0.001). These observations support the suggestion that chronic hyperglycaemia can influence the generation of free radicals, which may lead ultimately to increased lipid peroxidation and depletion of antioxidants, and thereby enhanced oxidative stress in subjects with type 2 diabetes mellitus.

Serum cystatin C, enzymuria, tubular proteinuria and early renal insult in type 2 diabetes.

This study investigates the association between serum cystatin C, serum creatinine concentrations, N-acetyl-beta-D-glucosaminidase (NAG enzymuria), urine alpha1-microglobulin (alpha1-MG) and beta2-microglobulin (beta2-MG) levels in subjects with type 2 diabetes (n=40, 20M/20F, age range 25-65 years; duration of diabetes 8-10 years) and age- and gender-matched healthy controls (n= 20). Exclusion criteria were absence of gross proteinuria, hypertension, dyslipidaemia or cardiovascular disease. Fasting blood samples and mid-stream specimen of urine (MSSU) were collected and serum creatinine, cystatin C, urine creatinine, NAG enzymuria, alpha1-MG and beta2-MG were measured. Diabetic subjects were separated into two groups based on albumin:creatinine concentration ratio. Group A: <3.5 (mg/mmol creatinine), group B: 3.5-35 (mg/mmol creatinine). While serum creatinine concentrations remained within the laboratory reference range for all groups, serum cystatin C concentration (mg/L) was significantly increased in group B (1.79 +/- 0.42 [mean +/- SD] compared to both control [0.81 +/- 0.10] and group A values [0.95 +/- 0.10]; both P<0.001). NAG enzymuria (units/mmol creatinine) was increased in both diabetic groups compared to control values (group B: 122 +/- 7, group A: 70 +/- 5, controls 27 +/- 2, all P<0.001). alpha1-microglobulin (microg/mmol creatinine) concentrations, similar in both the control group and group A diabetics at 1.10 +/- 0.10 and 1.11 +/- 0.21, respectively, were significantly elevated in group B at 2.10 +/- 0.41 (both P<0.01). Similarly, elevated beta2-MG (microg/mmol creatinine) levels were also observed in group B compared to both group A and control values (3.20 +/- 0.21 vs. 1.80 +/- 0.51 and 0.91 +/- 0.11, respectively; both P<0.001). In addition, group B levels were significantly higher than group A (P<0.001). These observations suggest that serum cystatin C is a more appropriate and effective biomarker for the overall estimation of GFR than serum creatinine values. In addition, increased serum cystatin C values were also associated with early renal tubular insult in subjects with type 2 diabetes, as characterised by increased NAG enzymuria, alpha1- and beta2-microglobulin excretion.

Urinary enzyme measurements as early indicators of renal insult in type 2 diabetes.

The association between urine microalbumin, alpha1-microglobulin concentration (alpha1MG) and the urinary enzyme activities of alanine aminopeptidase (AAP), N-acetyl-beta-D-glucosaminidase (NAG), alpha-glutathione-S-transferase (alphaGST) and pi-glutathione-S-transferase (piGST) is investigated in 36 type 2 diabetic and 15 age- and sex-matched non-diabetic subjects. Diabetic subjects were grouped into those with microalbuminuria <3 mg/L (group A: 7M/5F), 3-30 mg/L (group B: 5M/7F) and 30-300 mg/L (group C: 6M/6F). While serum creatinine concentration remained within the laboratory reference range (<115 mmol/L) in all experimental groups, alpha1MG excretion increased with the severity of microalbuminuria (control group and groups A, B and C mean [SD] values were 1.3 [0.21], 1.6 [0.11], 2.18 [0.42] and 2.8 [0.51] mg/mmol urinary creatinine, respectively). Activities of NAG (U/mmol creatinine) were significantly elevated in groups A, B and C at 98.7 (8.6), 112.8 (12.9) and 147.4(16.2), respectively, compared with the reference range <35 U/mmol creatinine (group C vs. groups A and B: P < 0.01). Activity of AAP (U/mmol creatinine) was significantly elevated in groups B and C at 7.6 (0.5) and 7.9 (0.6), respectively (both P < 0.001), compared to the control and group A values (2.5 [0.2]). Activity of piGST (U/mmol creatinine) was elevated in groups B and C at 2.6 (0.4) and 2.8 (0.5), respectively (both P < 0.001), compared to the control and group A values (1.1 [0.1]). Similarly, urine piGST activity was also elevated in groups B and C at 2.9 (0.6) and 3.1 (0.5), respectively (both P < 0.001), compared to control and group A values (1.3 [0.1] and 1.4 [0.2]). These results suggests that site-specific urinary biochemical markers provide valuable information about early renal proximal and distal tubular insult that ultimately may precede enhanced glomerular permeability in subjects with type 2 diabetes.

A prospective, comparative study of the para-aminobenzoic acid test and faecal elastase 1 in the assessment of exocrine pancreatic function.

BACKGROUND: The assessment of exocrine pancreatic insufficiency is part of the routine work-up of patients with persistent diarrhoea or suspected steatorrhoea. Direct and indirect tests for the diagnosis of exocrine pancreatic insufficiency have their drawbacks. Measurement of faecal elastase 1 by enzyme-linked immunoabsorbent assay is a simple, non-invasive, robust test for exocrine pancreatic insufficiency. METHODS: We performed a prospective comparison of the para-aminobenzoic acid test and faecal elastase 1 test in 45 patients being investigated for diarrhoea or suspected steatorrhoea. Details of clinical suspicion, imaging and response to treatment were recorded. RESULTS: Exocrine pancreatic function was normal in 20 patients with normal para-aminobenzoic acid and faecal elastase 1 levels. Eight patients had exocrine pancreatic insufficiency with low para-aminobenzoic acid and faecal elastase 1 levels, which improved with enzyme supplementation. In 14 of the 15 patients with low or borderline low para-aminobenzoic acid and normal faecal elastase 1 levels, a non-pancreatic cause was found; one patient had a false positive para-aminobenzoic acid test. Two had normal para-aminobenzoic acid but low faecal elastase 1 levels. One improved with pancreatic supplementation, and imaging revealed chronic pancreatitis. The other had a false positive faecal elastase 1 test related to profuse diarrhoea. CONCLUSIONS: Faecal elastase 1 estimation is a simple, non-invasive, robust test of exocrine pancreatic insufficiency, performed on an out-patient stool sample. Its diagnostic performance is superior to that of the para-aminobenzoic acid test in investigating patients with diarrhoea or suspected steatorrhoea.

Positive screening tests for barbiturates in urine samples in the York area over a 1-year period.

Following the screening of urine samples for a panel of drugs over a period of 1 year, a peak was observed during December 1999 and January 2000 in the numbers of samples testing positive for both morphine and barbiturates. This increase may be due to the adulteration of street heroin with barbiturate during this period.

Artefactual serum hyperkalaemia and hypercalcaemia in essential thrombocythaemia.

AIM: To investigate possible abnormalities of serum potassium and calcium levels in patients with essential thrombocythaemia and significant thrombocytosis. METHODS: 24 cases of essential thrombocythaemia with significant thrombocytosis (platelet count > 700 x 10(9)/litre) had serum potassium and calcium estimations performed at the time of maximum thrombocytosis before treatment, and at the time of low platelet count after treatment with cytoreductive drugs. Selected patients were further investigated with plasma sampling and estimation of ionised calcium and parathyroid hormone. RESULTS: At the time of maximum thrombocytosis six patients had serum hyperkalaemia (> 5.5 mmol/litre) and five had serum hypercalcaemia (> 2.6 mmol/litre). Following treatment and reduction of the platelet count, hyperkalaemia resolved in all cases and hypercalcaemia in four of the five cases. Mean serum potassium and calcium concentrations were raised (p < 0.0001) at maximum thrombocytosis compared with the values when the platelet count was low. Serum potassium and calcium values were significantly correlated at all stages. Measurements on plasma consistently corrected the hyperkalaemia but not the hypercalcaemia. Serum hypercalcaemia was associated with raised ionised calcium and normal parathyroid hormone concentrations. CONCLUSIONS: Essential thrombocythaemia with significant thrombocytosis is associated with serum hyperkalaemia and hypercalcaemia. The probable mechanism of hypercalcaemia is the secretion of calcium in vitro from an excessive number of abnormally activated platelets. It is thus likely that the hypercalcaemia is an artefact, as is the hyperkalaemia.

24-hour intragastric pH measurement in the assessment of duodenogastric reflux.

In this study we compared the results of measurement of duodenogastric reflux using 24-hour intragastric pH monitoring and nasogastric aspiration with analysis of bile acid and alpha amylase concentrations. Eight patients were studied at least 5 months after biliary and/or gastric surgery. The correlation coefficient of pH and bile acid concentration was 0.36 (p less than 0.001) and of pH and amylase concentration was 0.48 (p less than 0.001). If a pH greater than 4.0 was taken to indicate duodenogastric reflux this had a sensitivity of 84% compared with either high bile acid or amylase concentration. Bile acid concentration has been the "standard" method of measuring duodenogastric reflux. pH monitoring is relatively simple to perform, causes little patient distress, and can measure episodes of duodenogastric reflux over a continuous 24-hour period.

Osteoclast inhibition by pamidronate in metastatic prostate cancer: a preliminary study.

Twenty five hormone manipulated patients with prostate cancer and metastatic bone disease, treated at least 6/12 previously by hormone manipulation, were given intravenous infusions of Disodium Pamidronate (APD) over a 6 month period. Patients received 30 mg weekly for 4 weeks then twice monthly for 5 months. No other treatment was administered during study. Eleven of 17 patients with pain at the start of the study were pain free at the end. Fasting morning calcium excretion and serum osteocalcin fell significantly with Pamidronate (P less than 0.0001) and urine hydroxyproline was lowered in 13/20 evaluable patients at 6 months. Alkaline phosphatase fell in a proportion of patients and five of 17 patients with previously progressive bone scans stabilised (4) or regressed (1) on treatment. Rising acid phosphatase levels were also lowered in five patients. It is concluded that Pamidronate may be effective in palliating bone pain in some patients and has a stabilising influence on abnormally high bone turnover in metastatic prostate cancer. Further controlled studies of the compound are now warranted.

Simple, optimized liquid-chromatographic method for measuring total hydroxyproline in urine evaluated.

We have optimized a method for measuring total hydroxyproline (HYP) in urine by HPLC after release from urinary peptides by solid-phase hydrolysis on Dowex 50W x 8 ion-exchange resin. The HYP was derivatized with 4-chlor-7-nitrobenzo-2-oxa-1,3-diazole, and excess reagent was removed with the use of a 100-mg C18 Bond-Elut cartridge. The HYP derivative was separated isocratically at 30 degrees C on a 250 x 4.6 mm reversed-phase column containing 5-microns particles of Spherisorb S5 ODS-2, with S-carboxymethylcysteine as internal standard. Total assay time was 14 min. The standard curve for the method was linear from the detection limit for HYP, 3.6 mumol/L, to 10 mmol/L. The between-batch CV was less than 5.1% and the mean analytical recovery of HYP was 95% +/- 1.4%. Comparison with a commercially available colorimetric method showed good correlation: y = 1.158x + 19.76 mumol/L (Syx = 74, n = 120), but HPLC results were 15% higher, probably from incomplete hydrolysis with the colorimetric method. This method offers a considerable improvement in assay time, specificity, sensitivity, precision, and cost compared with the colorimetric method.

Serum carnitine levels in normal individuals.

Serum carnitine levels have been measured in 178 samples from 75 normal volunteers. We report a wide range of values (10-70 mumol/liter and 8-74 mumol/liter for free and acetylated carnitine, respectively) and a distinct difference between the ranges for males and females (p less than 0.001). There is also substantial, seemingly random fluctuation in any one individual's levels, when measured serially over several weeks.

Reassessment of faecal alpha-1-antitrypsin excretion for use as screening test for intestinal protein loss.

Faecal alpha-1-antitrypsin and 51Cr-albumin losses in 42 patients with either gastrointestinal or hepatic disease were compared. The reference range was derived from measurements in 20 controls without gastrointestinal disease. Alpha-1-antitrypsin excretion was increased in patients with excessive 51Cr-albumin loss, and correlations were found between alpha-1-antitrypsin clearance and 51Cr-albumin excretion. Because of the considerable overlap of faecal alpha-1-antitrypsin excretion between controls and patients, sensitivity and specificity of the test were only 58% and 80%, respectively. This poor reliability could not be explained by sampling error or temporal variations in alpha-1-antitrypsin excretion. These results show that although faecal alpha-1-antitrypsin excretion correlates with 51Cr-albumin excretion when whole groups of patients are studied, its poor sensitivity makes it an unreliable measure of enteric protein loss.

Essential trace element provision to patients receiving home intravenous nutrition in the United Kingdom.

The methods of provision of essential trace elements to patients receiving long-term home intravenous nutrition in the U.K. have been reviewed and their trace element status has been assessed. Over a 2 year period, 57 patients based on 15 hospitals throughout the country were studied. Although biochemical abnormalities of trace element status were frequent, related clinical complications were apparently rare. Zinc provision from commercial preparations generally required further supplementation. Copper requirements were variable. Manganese provision from trace element mixtures and chromium provision from contaminants of other parenteral nutrient solutions were excessive in many cases. Aluminium contamination is not significant in the nutrient solutions currently used in the U.K. The most common depletion state is for selenium which should probably be provided on a routine basis. The clinical consequences of long-term under- or over-provision of trace elements by the intravenous route requires further study.

Comparison of clinical scores and acute phase proteins in the assessment of acute Crohn's disease.

Fifty-two patients with Crohn's disease have been assessed using acute phase proteins and a scoring system. Thirty-nine underwent operation and intra-abdominal abscesses were found in seventeen. Both scores and acute phase proteins have been shown to reflect inflammation due to secondary sepsis in addition to that from active Crohn's disease. The scores were higher, and acute phase changes greater, in patients with sepsis than those without. By choosing a threshold for each variable that excludes patients without sepsis it has been found that a score greater than 181, ESR greater than 45 mm/h, CRP greater than 33 mg/l, orosomucoid greater than 1.8 g/l and albumin less than 26.7 g/l identify sepsis with a specificity greater than 95 per cent and sensitivity greater than 35 per cent. Over 70 per cent of patients with abscesses exceeded one or more of these thresholds. We believe that operative management should be strongly considered if one or more of these criteria are positive as such patients have a greater than 90 per cent chance of having an intra-abdominal abscess. This will prevent these abscesses eroding into adjacent viscera or to the surface with resulting fistula formation.

Intracellular free amino acids in undernourished patients with or without sepsis.

1. Thigh muscle samples for analysis were obtained from 35 control subjects and 12 severely ill patients, of whom five had disease-induced undernutrition and seven had associated sepsis. An improvement in the Bergström technique for analysing muscle needle biopsy samples was used. 2. Muscle water, chloride and DNA were increased in both groups of ill patients, indicating an increase in the extracellular component and loss of muscle protein. 3. Falls in the concentrations of glutamine, glutamate, arginine, histidine, ornithine and lysine and rises in those of glycine, serine, asparagine, tyrosine, valine, leucine, isoleucine, phenylalanine, methionine and tryptophan were observed in the group of ill patients compared with those in the control subjects. The changes were present mainly in the patients with sepsis, significant differences between these and the undernourished ones without associated sepsis being observed in glutamine, lysine, tyrosine, valine, leucine, isoleucine, phenylalanine and methionine. The changes were either absent or slight in plasma. 4. The increased concentrations observed in six essential amino acids in the septic patients, but not in those with undernutrition, is consistent with the increased catabolic rate of sepsis. 5. However, the branched-chain amino acids, and lysine, threonine, arginine, and histidine, were present in lower concentrations than might be expected from catabolism, presumably due to utilization. It is suggested therefore that septic patients might benefit from increased proportions of these amino acids in their feeding regimen.

Tinnitus in a patient with beta-thalassaemia intermedia on long-term treatment with desferrioxamine.

A women with beta-thalassemia intermedia, and iron overload following many years' treatment with oral iron, was treated with subcutaneous desferrioxamine. During the course of this treatment she developed tinnitus, which was considered to be a rare complication of the use of this chelating agent. The mechanism of this effect is not understood.

N tau-Methylhistidine excretion and myofibrillar protein breakdown in patients receiving intravenous or enteral nutrition.

1. N tau-Methylhistidine, nitrogen and creatinine were measured in the urine of 10 volunteers on normal and meat-free diets and in 10 vegetarians, and compared with the results from the urine of eight patients with intestine fistulae on intravenous or enteral nutrition containing no meat. The values obtained were used to calculate fractional breakdown rate of myofibrillar protein. 2. There was a significant fall in the excretion of N tau-methylhistidine and creatinine and in apparent fractional breakdown rates after 2 days on a meat-free diet. 3. One of the patients had lower, and two of the patients had higher, fractional breakdown rates compared with the vegetarians. 4. N tau-Methylhistidine and creatinine excretion may be a useful and non-invasive measurement of myofibrillar protein degradation in patients on meat-free diets. Firm conclusions cannot, however, be drawn without confirmatory, direct measurement of the breakdown rates of muscle protein in vivo.