Nucleotide variation in the mitochondrial genome provides evidence for dual routes of postglacial recolonization and genetic recombination in the northeastern brook trout (Salvelinus fontinalis).

Levels and patterns of mitochondrial DNA (mtDNA) variation were examined to investigate the population structure and possible routes of postglacial recolonization of the world's northernmost native populations of brook trout (Salvelinus fontinalis), which are found in Labrador, Canada. We analyzed the sequence diversity of a 1960-bp portion of the mitochondrial genome (NADH dehydrogenase 1 gene and part of cytochrome oxidase 1) of 126 fish from 32 lakes distributed throughout seven regions of northeastern Canada. These populations were found to have low levels of mtDNA diversity, a characteristic trait of populations at northern extremes, with significant structuring at the level of the watershed. Upon comparison of northeastern brook trout sequences to the publicly available brook trout whole mitochondrial genome (GenBank AF154850), we infer that the GenBank sequence is from a fish whose mtDNA has recombined with that of Arctic charr (S. alpinus). The haplotype distribution provides evidence of two different postglacial founding groups contributing to present-day brook trout populations in the northernmost part of their range; the evolution of the majority of the haplotypes coincides with the timing of glacier retreat from Labrador. Our results exemplify the strong influence that historical processes such as glaciations have had on shaping the current genetic structure of northern species such as the brook trout.

Development of an outpatient finger-prick glomerular filtration rate procedure suitable for epidemiological studies.

Development of an outpatient finger-prick glomerular filtration rate (GFR) procedure suitable for epidemiological studies. In clinical practice, reference GFR procedures are rarely used; in large-scale research studies, a great deal of effort and experience is required to obtain them, which is a considerable disincentive to using GFR as an end point. The major problem for both clinical staff and the subject is the length of time that the procedure takes, requiring continuous attendance in the outpatient clinic or its vicinity. Using iohexol as a marker, we therefore propose an alternative approach, which addresses this fundamental deterrent to a more widespread use of GFR measurement. Eighty-two GFR measurements were performed in a mixture of healthy subjects and patients with differing degrees of renal impairment with a wide range of GFRs. Serum was obtained from blood samples to enable a reference GFR to be calculated. Blood spots were collected on filter paper at the same intervals (120, 180, and 240 min), allowed to dry, and then sent through the post. Serum and blood spots were analyzed simultaneously for each individual by automated reverse-phase high-pressure liquid chromatography. Standard linear regression analyses confirmed a good agreement (r2 = 0.953) between the iohexol serum GFR and iohexol blood spots GFR. Bland-Altman analysis confirmed that there was no concentration bias. Paired comparisons (Wilcoxon's paired signed rank test) showed no significant difference between the two measurements. Capillary sampling is simple, effective, and significantly reduces the time and costs of performing plasma clearance GFR measurements. This approach will make the GFR measurement more accessible for clinical practice and large-scale epidemiological studies may become feasible.

Hyperhomocysteinaemia in a young woman presenting with stroke, associated with methylene tetrahydrofolate reductase C677T homozygosity.

We present a case of ischaemic stroke in a 23-year-old woman, associated with homozygous methylene tetrahydrofolate reductase (MTHFR)-C677T and hyperhomocysteinaemia. Her other risk factors for stroke were ostium secundum atrial septal defect and use of oral contraceptives. This case illustrates the need to include plasma homocysteine (Hcy) measurement in investigations following stroke. In the presence of hyperhomocysteinaemia, the MTHFR genotype should be determined. If the index case has the polymorphism, then all first-degree relatives should also be investigated by measurement of plasma Hcy and determination of MTHFR genotype.

Hypercarotenaemia or hypercarotenoidaemia.

BACKGROUND: Serum carotenoids consist of a variety of different compounds. Xanthoderma may result from an increased concentration of any of the carotenoids. METHODS AND RESULTS: High-performance liquid chromatography of serum carotenoids shows that a normal chromatogram contains mainly beta-carotene, lutein and lycopene. Serum alpha- and beta-carotene, beta-cryptoxanthin, lycopene, lutein and zeaxanthin were found to be increased in the investigation of hypercarotenaemia. CONCLUSION: Patients presenting with possible xanthoderma should have a dietary history taken and serum sent for carotenoid analysis.

Computer measurement of oral movement in swallowing.

This article describes a computer program that automatically detects and tracks small metal markers affixed to a subject's tongue and teeth in fluoroscopic image sequences of swallowing. The program, written in Microsoft Visual C++ using Windows NT 4.0 and the SAVANT imaging toolkit, involves marker detection and marker tracking. Marker detection is done by template matching. A generic marker template was designed by extracting the grayvalues of pixels within an imaged marker. Template matching with a weighted center-of-mass calculation determined marker location with subpixel accuracy. Marker tracking employed a nearest-neighbor algorithm since (a) the movement of each marker was less than the distance between any two markers and (b) marker trajectories did not overlap. Effects of head motion were attenuated by computing tongue trajectories with respect to a constant frame of reference given by reference markers on the maxillary teeth. Motions were converted from pixels/frame to millimeters/second using a calibration ring secured to the subject's neck. Results for several image sequences showed that our program performs very well in terms of marker detection and trajectory determination. Comparison of automatic and manual tracking of the same image sequences indicated a high degree of correspondence. Automatic tracking of oral movement by computer is a useful tool in kinematic studies of swallowing.

The role of ventromedial prefrontal cortex in the recovery of extinguished fear.

Conditioned fear responses to a tone paired with footshock extinguish when the tone is presented repeatedly in the absence of shock. Rather than erase the tone-shock association, extinction is thought to involve new learning accompanied by inhibition of conditioned responding. Despite much interest in extinction from a clinical perspective, little is known about the neural circuits that are involved. Although the prefrontal cortex has a well established role in the inhibition of inappropriate behaviors, previous reports have disagreed as to the role of the ventromedial prefrontal cortex (vmPFC) in extinction. We have reexamined the effects of electrolytic vmPFC lesions made before training on the acquisition, extinction, and recovery of conditioned fear responses in a 2 d experiment. On Day 1 vmPFC lesions had no effect on acquisition or extinction of conditioned freezing and suppression of bar pressing. On Day 2 sham rats recovered only 27% of their acquired freezing, whereas vmPFC-lesioned rats recovered 86%, which was indistinguishable from a control group that never received extinction. The high recovery in lesioned rats could not be attributed to decreased motivation or altered sensitivity to footshock. vmPFC lesions that spared the caudal infralimbic (IL) nucleus had no effect. Thus, the vmPFC (particularly the IL nucleus) is not necessary for expression of extinction, but it is necessary for the recall of extinction learning after a long delay. These data suggest a role of the vmPFC in consolidation of extinction learning or the recall of contexts in which extinction took place.

Long-term effects of naltrexone on self-injurious behavior.

A subgroup of self-injuring patients responds positively to the opiate-blocking agent naltrexone in acute, double-blind studies. In this study we examined the effects of naltrexone after acute treatment and the long-term effects of naltrexone on SIB. Rates of SIB were collected from pretreatment baseline; a second baseline a year after the acute trial; and a subsequent 12-month double-blind, placebo-controlled treatment. A subgroup of patients decreased SIB for a year without treatment after acute exposure to naltrexone. Five participants who decreased SIB by 70% after acute treatment increased SIB to the long-term treatment with naltrexone. In contrast, those for whom SIB increased over the one-year treatment hiatus decreased their SIB after the first long-term treatment. Discussion of these complex effects considered the role of background opioid levels, dosing, and treatment regimen of naltrexone and other factors limiting receptor adaptation among patients who exhibit SIB.

Long-term glycaemic control directly correlates with glomerular filtration rate in early Type 1 diabetes mellitus before the onset of microalbuminuria.

Hyperfiltration occurs early in diabetes mellitus and has been implicated in the development of microalbuminuria. Our aim was to re-examine the controversial relationship between glycaemic control and glomerular filtration (GFR) in normoalbuminuric, normotensive, non-obese patients with short duration Type 1 diabetes mellitus (DM). We studied 75 Type 1 DM patients, 35 male, aged 18-42 years, with a duration of diabetes of 4-8 years. GFR was determined by inulin clearance; hyperfiltration was defined as above 145 ml min(-1) 1.73 m(-2) (equivalent to 2 SD above mean for a control population). Analysis was by paired Student's t-testing and linear regression. GFR correlated significantly with HbA1c (r= 0.47, p < 0.0001) and fructosamine (r= 0.24, p = 0.035). Mean HbA1c and fructosamine in the 13 patients with hyperfiltration was significantly higher than in the rest of the group (HbA1c: 9.2% (95% C.I. 7.9-10.4%) vs 7.6 % (7.2-7.9), p= 0.002; fructosamine: 479 micromol l(-1) (450-507) vs 410 micromol l(-1) (388-432), p = 0.009. This significant difference persisted even when the two highest values of HbA1c or fructosamine were removed from analysis. Effective renal plasma flow, assessed by PAH clearance, also correlated in all patients with HbA1c (r=0.31, p=0.039). We conclude that poor glycaemic control directly correlates with hyperfiltration and renal hyperperfusion in early Type 1 DM.

Serotonin and heart rate in hypertensive and normotensive subjects.

Plasma and platelet levels of 5-hydroxytryptamine (5 HT) may be altered in essential hypertension. To establish the determinants and correlates of 5 HT in plasma and platelets, we studied 53 untreated subjects with essential hypertension (26 men; 30 whites; mean supine blood pressure 172/101 mm Hg; mean age 49.3 +/- 1.5 years) and 61 normotensive subjects (37 men; 47 whites; mean supine blood pressure 128/78 mm Hg; mean age 42.8 +/- 1.6 years). Plasma and platelet 5 HT were assayed by reverse-phase high performance liquid chromatography with electrochemical detection. No significant difference was found in platelet-poor plasma or platelet 5 HT levels in hypertensive or normotensive subjects (plasma: 43.0 +/- 4.2 and 39.6 +/- 4.4 nmol/L; platelet: 1.65 +/- 1.22 and 1.70 +/- 1.39 nmol/10(9) cells in hypertensive and normotensive subjects, respectively). No significant correlation was found between plasma or platelet 5 HT and systolic or diastolic blood pressure (plasma: r = 0.01 and 0.01 in normotensive subjects and r = 0.01 and -0.14 in hypertensive subjects; platelet: r = 0.12 and 0.13 in normotensive subjects and r = 0.02 and -0.09 in hypertensive subjects). However, plasma 5 HT was associated with supine and standing pulse rates (supine: r = 0.27, p = 0.05 in normotensive subjects and r = 0.54, p < 0.001 in hypertensive subjects; standing: r = 0.19 and r = 0.46, p < 0.001, respectively). Significant relations were also found between platelet 5 HT levels and supine and standing heart rate in the subjects mentioned above (supine: r = 0.28, p = 0.05 in normotensive subjects and r = 0.64, p < 0.001 in hypertensive subjects; standing: r = 0.24 and r = 0.51, p < 0.001, respectively). These associations were stronger in the hypertensive group as a whole, and they held when adjustment was made for differences in age and total blood cholesterol. The present study showed that plasma and platelet 5 HT are not significantly altered in hypertensive subjects. However, plasma and platelet 5 HT levels showed a significant association with supine and standing pulse rate predominantly in hypertensive subjects. This is consistent with experimental evidence of a positive chronotropic effect of 5 HT on perfused hearts and it suggests a possible role of plasma serotonin in the regulation of heart rate.

High-performance liquid chromatographic determination of pyridinium crosslinks in serum, urine and dialysate of patients in chronic renal failure.

This study describes an isocratic reversed phase high-performance liquid chromatographic method for the analysis of pyridinium crosslinks in serum, urine and dialysates obtained from patients with chronic renal failure on haemodialysis. The mean (SD) urine pyridinoline (PYD) and deoxypyridinoline (DPD) to creatinine (Cr) ratio in 19 healthy volunteers was 28.9 (6.3) and 9.1 (3.6) mumol/mol, respectively. In the 22 patients the PYD/Cr and DPD/Cr ratio was 244.6 (436.5) and 66.5 (116.8) mumol/mol, respectively. The mean serum PYD concentration in 29 patients of 268.5 (334.4) nmol/L was significantly higher than that of 5.9 (1.5) nmol/L found in normal volunteers: the mean DPD concentration was 82.9 (93.7) nmol/L in the patients but was undetectable in the serum from the normal volunteers. The concentration of crosslinks in pre-dialysis serum samples was higher than those found post-dialysis reflecting a significant removal of the crosslinks during dialysis. The assay of pyridinium crosslinks in serum, urine and dialyses fluid could potentially provide evidence of bone collagen turnover in patients in renal failure. Their measurement in serum and dialysate could be particularly useful in anuric patients.

Pyridinium crosslinks in patients on haemodialysis and continuous ambulatory peritoneal dialysis.

BACKGROUND: The urine excretion of the pyridinium crosslinks of collagen, pyridinoline (PYD) and deoxypyridinoline (DPD) closely reflect bone resorption and their assay has been used as specific markers of mature collagen turnover. The aims of this study were to evaluate the use of these markers to predict the severity of osteodystrophy in patients with chronic renal failure. METHODS: Using an isocratic ion-paired reverse-phase high-performance liquid chromatography, PYD and DPD were determined in the serum, urine and dialysate of 48 patients with chronic renal failure undergoing haemodialysis (n = 28) or continuous ambulatory peritoneal dialysis (n = 20). Nineteen apparently healthy subjects were studied as controls. RESULTS: In all groups, serum and urine crosslinks excretion showed poor correlation with age. In the patients urine PYD/creatinine and DPD/creatinine were significantly (P < or = 0.03 and < or = 0.001 respectively) higher than normal; urine PYD and DPD levels were highly correlated with each other (r = 0.98) and with serum PTH (r = 0.84 and 0.83 respectively). The mean (SD) predialysis serum PYD, 269 (334) nmol/l, was significantly (P < or = 0.003) elevated compared with normal patients, 4.1 (0.6) and pre-dialysis serum DPD was 82.9 (93.7) nmol/l. DPD was below the detection limit of the assay in normal sera. In the patients postdialysis decreases in serum PYD and DPD were statistically significant (P < 0.0002 and P < 0.0007 respectively). PYD and DPD were found in the dialysate of patients on haemodialysis as well as 24-h dialysate in patients on CAPD. Dialysate PYD and DPD were highly correlated with each other (r = 0.80) and with dialysate creatinine (r = 0.76 and r = 0.62 respectively). In the patients, the mean serum, urine and dialysate PYD and DPD increased with the duration on dialysis. These findings confirm that metabolic bone disease increases in patients with duration of chronic renal failure. CONCLUSION: Estimation of serum crosslinks levels has potential as an additional tool in the diagnosis and monitoring of renal osteodystrophy. The ability to determine crosslink levels in serum and dialysate should be particularly useful in patients who are unable to produce urine.

An automated enzymatic inulin assay, capable of full sinistrin hydrolysis.

Renal inulin clearance remains the standard by which other methods of measuring glomerular filtration rate are judged. A fully automated enzymatic assay capable of use with linear configuration inulin was recently published (Summerfield AL, et al. Clin Chem 1993; 39:2333-7). Sinistrin, a readily soluble preparation of polyfructan with side branching, is more suitable for clinical use and far more widely used in Europe. By modifying the incubation phase of samples with inulinase, incorporating a kinetic modification to the method of fructose analysis, and increasing the buffer strengths, we report a fully automated system, with minimal sample prehandling capable of complete sinistrin hydrolysis, and adapted for use on the Cobas Mira.

Thyroid antoantibodies and the response to thyrotropin releasing hormone in patients with subclinical hypothyroidism.

AIM: To evaluate the clinical usefulness of the thyrotropin releasing hormone (TRH) test and estimation of thyroid autoantibody concentrations in patients with borderline raised thyroid stimulating hormone (TSH). METHODS: The records of 34 consecutive patients with persistent borderline increased TSH (4.4-9.9 mU/l) referred to the Medical Investigation Unit were reviewed. The response of patients with thyroid autoantibodies to the TRH test was compared with that of patients with a negative antibody screen. RESULTS: Eleven (44%) of 25 patients with positive anti-thyroid microsomal and/or thyroglobulin antibody tests and three (33%) of nine patients with a negative antibody screen had hypothyroid responses to TRH. Neither age nor sex affected the response to TRH. Basal TSH alone was poorly correlated with these indices. Twelve (35%) patients who had elevated basal TSH had a normal response to the TRH test. CONCLUSION: Patients with positive or negative thyroid autoantibodies and an exaggerated response to the TRH test should be regarded as hypothyroid and treated with thyroxine. Patients with positive thyroid autoantibodies and normal TSH response may subsequently develop hypothyroidism and should be given long term follow up.

A comparison of overnight and 24 hour collection to measure urinary catecholamines.

The period of urine collection used to measure excretion of catecholamines varies in epidemiological practice. We set out to compare overnight with 24 hour collection. Twenty-four subjects each collected urine for 24 hours, with the overnight urine being separately collected. The correlation of overnight and 24 hour catecholamines was highest when both measures were standardised for creatinine excretion and when creatinine excretion was adjusted for urine flow rate. The observed correlations were 0.74 for dopamine, 0.81 for noradrenaline and 0.54 for adrenaline. The use of overnight collections may therefore require a sample size up to 1.5 times as large (for noradrenaline) or 3.4 times as large (for adrenaline) to achieve the same power as with 24 hour collections. However, the figures given exaggerate the advantage of 24 hour collections if these incorporate measurement errors that are not present in overnight collections.

Memory deficits associated with chronic fatigue immune dysfunction syndrome.

Performance on tests of memory in 39 patients who met Center for Disease Control (CDC) criteria for chronic fatigue immune dysfunction syndrome (CFIDS) was compared with 23 depressed patients (DSM-III-R) and 129 healthy controls. Although the CFIDS patients had normal neuropsychological profiles, they significantly overestimated their ability (metamemory), performed significantly worse on tests of recall as context increased (e.g., recognition), made more errors when rehearsal was prevented, and had delayed mental scanning as memory load increased. The overall pattern indicated that CFIDS patients had a significant memory deficit, far worse than implied by CDC criteria. The pattern for CFIDS patients was consistent with temporal-limbic dysfunction and significantly different than depressed patients and control subjects.