A new method for the determination of L-dopa and 3-O-methyldopa in plasma and cerebrospinal fluid using gas chromatography and electron capture negative ion mass spectrometry

L-3-(3,4-Dihydroxyphenyl)alanine (DOPA) and its 3-O-methyl metabolite (OMD) were measured in plasma and cerebrospinal fluid by a new assay which combines N,O-acetylation of amino acids in aqueous media, preparation of pentafluorobenzyl esters under anhydrous conditions, and analysis by gas chromatography-electron capture negative ion mass spectrometry. The N,O-acetyl, carboxy-PFB derivatives gave abundant carboxylate anions ([M-CH2C6F5]-) which were suitable for sensitive analysis using selected ion monitoring. Plasma and CSF samples were sufficiently purified by a simple organic solvent extraction. Analytical recovery for DOPA was 100.2 +/- 3.7% at the level of 100 nmol/l. Analysis of DOPA in plasma was performed with a relative standard deviation of 5%. The limit of quantitation in plasma and CSF was at the sub-nmol/l level. In healthy adults, DOPA concentration in plasma was 9.0 +/- 2 nmol/l (n = 11) and in CSF 3.5 +/- 0.9 nmol/l (n = 9). The concentration of OMD in plasma was 99.1 nmol/l (pool of 24 samples) and 15.3 nmol/l in CSF (pool of 12 samples). Measurement of 5-[2H]DOPA and 5-[2H]OMD in plasma of a healthy individual who had been orally loaded with 3,5-[2H2]tyrosine (150 mg kg body wt) was possible for several hours after the load.     

delta-Aminolaevulinic acid in plasma, cerebrospinal fluid, saliva and erythrocytes: studies in normal, uraemic and porphyric subjects

delta-Aminolaevulinic acid (ALA) was determined by g.l.c. with electron-capture detection. Normal plasma level was 92 nmol/l (SD = 39, n = 89, range 24-270 nmol/l). ALA was undetectable in 35 of 53 samples of normal cerebrospinal fluid (limit of assay 2 nmol/l). The mean of the other 18 samples was 19 nmol/l (SD = 10, range 6-36 nmol/l). Salivary ALA was generally only 10-30% of the plasma level in normal and porphyric subjects. Erythrocytes of normal and porphyric subjects contained no detectable ALA and were impermeable to its entry. ALA clearance correlated closely with that of creatinine, consistent with it being excreted by glomerular filtration with limited tubular reabsorption. In chronic renal failure, serum ALA was elevated to a maximum of three to four times the normal, but its urinary excretion was reduced, in keeping with lessened production. In two cases of acute intermittent porphyria with overwhelming neuropathy the maximum plasma levels of ALA were 9 and 12 mumol/l. Haematin infusion decreased the ALA levels but without obvious clinical benefit. Limited neurological recovery occurred without major reduction in plasma levels of ALA. One subject's attack was precipitated by pregnancy. The neonate was apparently normal, despite high levels of ALA in maternal plasma throughout gestation and a high level of ALA in the cord blood. The observations described here do not support the view that ALA may be directly neurotoxic.     

Cerebrospinal fluid levels of catechols in patients with neurogenic orthostatic hypotension

In multiple system atrophy (MSA) and pure autonomic failure (PAF), orthostatic hypotension (OH) results from deficient noradrenaline release from sympathetic nerves during standing. Post-mortem findings have indicated loss of central noradrenergic cells in both diseases. The present study sought in vivo neurochemical evidence for central noradrenergic deficiency in patients with OH due to MSA or PAF. A total of 28 patients with OH (18 with MSA; 10 with PAF) had cerebrospinal fluid and blood sampled for levels of noradrenaline and its neuronal metabolite dihydroxyphenylglycol. A control group of 44 subjects included 10 elderly normal volunteers, 10 patients with Alzheimer's disease, 18 patients with dysautonomia (postural tachycardia syndrome or neurocardiogenic syncope) and six patients with MSA in the absence of OH. Patients with OH had lower cerebrospinal fluid concentrations of noradrenaline (0.53+/-0.07 nmol/l) and dihydroxyphenylglycol (6.52+/-0.46 nmol/l) than did control subjects (0.90+/-0.09 and 9.64+/-0.46 nmol/l respectively; P =0.0001). The MSA+OH group had higher plasma levels of both catechols (noradrenaline, 1.31+/-0.16 nmol/l; dihydroxyphenylglycol, 5.08+/-0.43 nmol/l) than did the PAF group (noradrenaline, 0.38+/-0.08 nmol/l; dihydroxyphenylglycol, 2.53+/-0.30 nmol/l; P <0.001), despite similarly low cerebrospinal fluid levels. Among MSA patients, those with OH had lower cerebrospinal fluid levels of noradrenaline and dihydroxyphenylglycol than those without OH (noradrenaline, 1.71+/-0.64 nmol/l; dihydroxyphenylglycol, 10.41+/-1.77 nmol/l respectively; P =0.006). The findings are consistent with central noradrenergic deficiency in both MSA+OH and PAF. In MSA, central noradrenergic deficiency seems to relate specifically to OH.     

Measurement of 5-aminolaevulinic acid by reversed phase HPLC and fluorescence detection

A new method for sensitive measurement of delta-aminolaevulinic acid (ALA) in biological material is described. ALA is derivatized with dansyl chloride, separated by HPLC and estimated using a fluorescence detector. The pretreatment of biological samples includes desamination of L-alpha-aminoacids with L-aminoacid-oxidase before dansylation. The sensitivity of the method is slightly below 1 pmol/injection for standards and the lower limit of quantification is 0.1 mumol/l for plasma and 10 nmol/l for cerebrospinal fluid. Reference values in plasma are 3.53 +/- 1.75 (SD) (n = 43) mumol/l and in packed erythrocytes they ranged from 6 to 26 mumol/l (mean: 14.0 +/- 5.5 mumol/l). In cerebrospinal fluid of non-porphyric individuals less than 2 nmol/l were recovered.     

Increased plasma dihydroxyphenylalanine during sympathetic activation in humans is related to increased norepinephrine turnover

Plasma concentrations of dihydroxyphenylalanine (DOPA) were measured before and during isometric handgrip exercise or mental stress and after coffee drinking or intravenous infusion of desipramine to examine the influence of sympathetic nervous activity on DOPA formation. Sympathetic activity was assessed by the spillover of norepinephrine into plasma. Turnover of norepinephrine was assessed by the plasma concentration of its intraneuronal metabolite, dihydroxyphenylglycol (DHPG). In normal subjects the resting plasma concentration of DOPA was 6.05 +/- 0.16 nmol/L (n = 42). Plasma DOPA level was increased by stimulation of the sympathetic nervous system; handgrip exercise caused a 0.49 +/- 0.07 nmol/L increase (n = 15), mental stress a 0.25 +/- 0.10 nmol/L increase (n = 34), and coffee drinking a 0.85 +/- 0.19 nmol/L increase (n = 9). Desipramine decreased plasma DOPA level by 0.25 +/- 0.06 nmol/L (n = 23). The small but consistent changes in plasma DOPA level during manipulations of sympathetic activity were positively correlated with changes in norepinephrine spillover (r = 0.55, n = 81) and plasma DHPG level (r = 0.66, n = 81). Percentage increases in plasma DOPA level during sympathetic activation were similar to those in plasma DHPG but were a sixth of the percentage increases in norepinephrine spillover. The similar increases in plasma DOPA and DHPG levels indicated that production of DOPA was related to the turnover of norepinephrine in sympathetic nerves. The smaller percentage increases in plasma DOPA (smaller than those in norepinephrine spillover) were consistent with the partial contribution of exocytotic neurotransmitter release to the turnover of norepinephrine in sympathetic nerves.     

Clinical relevance of the quantification of apolipoprotein E in cerebrospinal fluid

Apolipoprotein E was measured in paired sera and cerebrospinal fluid samples from 483 neurological patients. The average apolipoprotein E concentration was 7.5 (+/- 3.1) mg/l in cerebrospinal fluid and 93.5 (+/- 29.7) mg/l in serum. Mean apolipoprotein B concentrations in 88 patients were 0.77 +/- 3.4 mg/l in cerebrospinal fluid and 1.06 +/- 0.31 g/l in serum. The apolipoprotein E concentration in cerebrospinal fluid was much greater than expected for passive diffusion from serum. By contrast to apolipoprotein B, there was no correlation between the apolipoprotein E cerebrospinal fluid/serum concentration quotient and the albumin concentration quotient. This suggests that apolipoprotein E in cerebrospinal fluid, unlike apolipoprotein B, is locally synthesized and that the use of a cerebrospinal fluid/serum concentration quotient is unnecessary. In a control group (n = 64) the mean cerebrospinal fluid apolipoprotein E value (+/- SD) was 5.9 (1.6) mg/l. Elevated cerebrospinal fluid apolipoprotein E concentrations were observed in acute (n = 22, 12.5 +/- 6.2 mg/l) and chronic inflammatory central nervous system diseases (n = 15, 10.4 +/- 2.4 mg/l).     

Determination of plasma oxalate with oxalate oxidase

A method for the determination of plasma oxalate using oxalate oxidase (EC 1.2.3.4) and deproteinised plasma is described. Reference values are 1.2-6.4 mumol/l (n = 24, mean +/- SD 3.3 +/- 1.5 mumol/l). The sensitivity is 6-7 nmol, the accuracy 3-5 nmol, and the coefficient of variation 10.4% (at a level of 23 nmol). The recovery from plasma spiked with oxalate was 105 +/- 8% (n = 8). Allowing blood to stand for 4 h at room temperature had no effect on plasma oxalate levels; inhibitors of glyoxylate converting enzymes interfered with oxalate oxidase.     

Immunoradiometric assay for human complement component C9 utilising monoclonal antibodies

A two-site immunoradiometric assay for human C9 has been developed. The assay utilised two non-competing monoclonal antibodies to C9 in a single incubation assay protocol. The detection limit of the assay was 0.1 ng (1.4 X 10(-15) moles) in a sample volume of 100 microliters. Using this assay the C9 concentration in normal human plasma was 60.2 +/- 14.9 mg/l (mean +/- 1 standard deviation, 8.5 X 10(-10) mol/l. Significantly elevated levels were found in the plasma of patients with rheumatoid arthritis (90.4 +/- 19.9 mg/l, mean +/- 1 SD). Measurements of C9 in cerebrospinal fluid and synovial fluid were also performed. The low levels of C9 in cerebrospinal fluid (less than 1 mg/l), undetectable by previously available assay methods, were easily measurable with this highly sensitive assay.     

Plasma dihydroxyphenylalanine (DOPA) is independent of sympathetic activity in humans

To clarify the origin of plasma DOPA (3,4-Dihydroxyphenylalanine), the relationship between plasma DOPA and acute or chronic changes in sympathetic activity has been studied. Plasma DOPA and noradrenaline (NA) concentrations were measured by reverse-phase high-performance liquid chromatography with electrochemical detection. Administration of clonidine to healthy men decreased plasma NE markedly compared to no drug. Plasma DOPA decreased slightly but significantly with time, but values were identical after clonidine compared to no drug. Baseline plasma NE concentrations were significantly reduced in diabetic patients with autonomic neuropathy compared to diabetics without neuropathy, whereas baseline plasma DOPA concentrations were similar in the three groups investigated: 6.55 (5.03-7.26, median [interquartile range], n = 8) nmol l-1 in diabetics with neuropathy, 7.41 (5.79-7.97, n = 8) nmol l-1 in diabetics without neuropathy, and 6.85 (5.58-7.36, n = 8) nmol l-1 in controls. No relationship was obtained between baseline values of plasma NE and plasma DOPA. Plasma DOPA did not change in the upright position, whereas plasma NE increased significantly. Our results indicate that plasma DOPA is not related to sympathetic activity and may be of non-neuronal origin.     

Determining zinc coproporphyrin in maternal plasma--a new method for diagnosing amniotic fluid embolism.

We measured the concentration of zinc coproporphyrin I (ZnCP-I), a characteristic component of meconium, in maternal plasma by fluorometry after HPLC. We obtained plasma samples from 89 women: 35 at weeks 10-40 of normal pregnancy, 41 shortly after normal delivery, 4 from patients with amniotic fluid embolism (AFE), and 9 from non-AFE patients with intra- or postpartum shock caused by genital bleeding. The plasma ZnCP-I concentration was 97 (SD 83, range 38-240) nmol/L in the AFE patients, 11 (SD 9.2) nmol/L in the non-AFE patients, 12 (SD 7.9) nmol/L during normal pregnancy, and 26 (SD 10) nmol/L shortly after normal delivery. We suggest that measuring ZnCP-I in maternal plasma by fluorometry on HPLC is a rapid, noninvasive, and sensitive method for diagnosing AFE and propose 35 nmol/L as the cutoff value for the ZnCP-I concentration in maternal plasma for the diagnosis of AFE.     

Radioimmunoassay of testosterone not bound to sex-steroid-binding protein in plasma

To measure the concentration of testosterone (T) that is not bound to sex-steroid-binding protein (SBP) in plasma, we quantified by radioimmunoassay the T in the supernates of plasma samples after precipitation with 50%-saturated ammonium sulfate. The concentrations of non-SBP-bound T. directly measured with this assay, correlated significantly (P less than 0.001) with those deduced from measurement of the percentage of non-SBP-bound T determined with [3H]T as tracer or from mathematical models according to the law of mass action. It also correlated significantly with the ratio of T to SBP and with the concentration of nonbound T. As determined with this assay, the mean concentration of non-SBP-bound T in normal men was higher in young (4.67, SD 2.68 nmol/L; n = 30) than in older (greater than 40 years) subjects (2.48, SD 1.61 nmol/L; n = 35; P less than 0.001) and lower than normal in hyperthyroid (1.61, SD 0.91 nmol/L; P less than 0.01) or infertile men (3.28, SD 1.70 nmol/L; P less than 0.01). In women, non-SBP-bound T was higher in hirsute patients (0.24, SD 0.11 nmol/L; P less than 0.01) and was lower during pregnancy (0.09, SD 0.05 nmol/L; P less than 0.05) than in normal women during the follicular phase (0.16, SD 0.07 nmol/L). We conclude that this direct measurement of non-SBP-bound T in plasma is suitable for routine use and represents a reliable index of androgenicity in human pathology, particularly when alterations of the binding capacity of SBP modify the concentrations of total T.     

A radioimmunoassay for 18-hydroxycortisol in plasma and urine

Increased excretion of 18-hydroxycortisol has been proposed as a specific biochemical marker for differential diagnosis of primary aldosteronism. We describe the development of a direct RIA with an 125I label that permits measurement of the steroid in less than or equal to 0.5 microL of urine or less than or equal to 25 microL of plasma. For control subjects, the mean concentrations of 18-hydroxycortisol in urine and plasma were 310 (SD 178) nmol/24 h (n = 32) and 2.27 (SD 0.80) nmol/L (n = 37), respectively; patients with Conn's adenoma or glucocorticoid-remediable aldosteronism had values for urine in the range 1084 to 6534 nmol/24 h and concentrations in plasma ranging from 6.49 to 31.20 nmol/L. Patients with idiopathic zona glomerulosa hyperplasia had values for urine and plasma ranging from 353 to 734 nmol/24 h and from 0.26 to 6.60 nmol/L, respectively. Concentrations of 18-hydroxycortisol in urine clearly discriminate patients with idiopathic hyperplasia from those with other forms of primary aldosteronism, but further work is required to assess the diagnostic accuracy of determinations in plasma.     

Measurement of brain natriuretic peptide in plasma samples and cardiac tissue extracts by means of an immunoradiometric assay method

We evaluated the analytical characteristics and clinical usefulness of a commercial immunoradiometric assay (IRMA) kit for brain natriuretic peptide (BNP). Mean (+/-SD) plasma BNP concentrations measured in 129 normal subjects were 2.9+/-2.7 pmol/l (median 2.2 pmol/l; range 0.1-12.4 pmol/l). The mean (+/- SD) value observed in healthy men (2.1 +/- 2.0 pmol/l, n = 49) was significantly (p=0.0009) different to that found in women (3.4 +/- 2.9 pmol/l, n=80). A positive relationship (R=0.214, p=0.0174) was found between BNP values and age. In 65 patients with cardiac diseases, BNP levels increased with the progression of clinical severity of disease; patients with more severe disease [NYHA functional class III-IV, mean (+/- SD) BNP +/- 254 +/- 408 pmol/l, n=22] showed significantly (p<0.0001) increased values compared to patients with mild symptoms of disease (NYHA functional class I-II, mean (+/- SD) BNP=19.6 +/- 17.2 pmol/l, n=43). Furthermore, in 32 patients with chronic renal failure, greatly increased (p<0.0001) BNP values were found both before (mean +/- SD=88. 1+/- 111.1 pmol/l) and after haemodialysis (mean +/- SD=65.6 +/- 76.7 pmol/l), with a significant reduction after haemodialysis (p=0.0004) compared to pre-haemodialysis. The mean (+/- SD) BNP value found in atrial extracts collected during aorto-coronary bypass operations in 15 patients was 14.5 +/- 51.9 pmol/g of cardiac tissue. Moreover, the mean (+/- SD) tissue levels of BNP in 7 heart transplant recipients were 128.4 +/- 117.2 pmol/g of cardiac tissue in atrium, 68.4 +/- 76.7 pmol/g in ventricle, and 10.9 +/- 8.5 pmol/g in interventricular septum. Finally, BNP values found in cardiac tissues of two subjects collected at autopsy were considerably lower (on average 1/1000) than those observed in cardiac tissues of patients with cardiac diseases. The IRMA method for BNP determination evaluated in this study showed a good degree of sensitivity, precision and practicability. Therefore, this method should be a reliable tool for the measurement of plasma BNP levels for both experimental studies and routine assay.     

Determination of gangliosides and sulfatide in human cerebrospinal fluid with a microimmunoaffinity technique

An immunostaining procedure has been developed for the assay of the gangliotetraose gangliosides and sulfatide in cerebrospinal fluid. Gangliosides of the gangliotetraose series were individually determined with cholera toxin B-subunit (CT-B) and an anti CT-B monoclonal antibody after chromatography and sialidase hydrolysis to GM1 on high performance thin-layer plates. Sulfatide was determined by thin-layer chromatography using an anti-sulfatide antibody. The method was applied to normal cerebrospinal fluid from 20 adults and 30 children. The concentration of the gangliotetraose series gangliosides in adults varied from 100-300 nmol/l with a mean value of 230 +/- 56 nmol/l. Corresponding values for sulfatide were 30-225 nmol/l and 140 +/- 46 nmol/l. The values for gangliosides and sulfatide in children increased during development. The major gangliosides in cerebrospinal fluid of adults were GT1b and GD1b and in children GD1a and GT1b.     

gamma-Glutamylglutamine identified in plasma and cerebrospinal fluid from hyperammonaemic patients

gamma-Glutamylglutamine has been identified in plasma and cerebrospinal fluid. Preparative high voltage electrophoresis was used to isolate the compound from two to three ml of sample. Analysis of archival material from eight patients with urea cycle disorders showed that plasma gamma-glutamylglutamine was 20-214 mumol/l when the plasma glutamine was greater than 1,000 mumol/l and that the relationship was strongly positive (P less than 0.0005). Plasma gamma-glutamylglutamine concentration was also raised in one patient with secondary hyperammonaemia, but not in three other cases, one of whom had a plasma glutamine of 10,000 mumol/l. Cerebrospinal fluid from the last patient contained 97 mumol/l of gamma-glutamylglutamine. Samples of cerebrospinal fluid from two patients with urea cycle disorders were available and the gamma-glutamylglutamine levels were 203 and 313 mumol/l. gamma-Glutamylglutamine and 5-oxoproline concentrations were not significantly increased in urine from any of these patients.     

Quantitative Determination of Putrescine,Cadaverine, Spermine and Spermidixe

Plasma bone Gla protein (BGP) was determined by radio-immunoassay in 266 healthy adults, men (n=132) and women (n = 134), aged 20–79 years. In the women aged 30–69 years, plasma BGP increased significantly with age (r = 0.44. p<0.001). and a particularly steep increase was seen from 1.1 ± 0.5 (mean±1 SD) in the fifth decade to 2.0 ± 1.4 nmol/l in the seventh decade. In men, aged 30–69 years, no correlation was found between plasma BGP and age (r= ?0.07, NS). Plasma bone Gla protein is removed from the circulation mainly by the kidneys and the increased plasma BGP in the women could be caused by decreased renal clearance. The interrelationship was analysed by means of partial correlation. When creatinine clearance was held constant in women, BGP still correlated positively with age (r = 0.40. p<0.001). but not with creatinine clearance (r=0.003, NS) when age was fixed. Plasma BGP was significantly increased above normal in 35 patients with chronic renal failure (10.2±14.6 nmol/l). Non-linear regression analysis showed that plasma BGP was within the normal range when 24-h creatinine clearance was greater than 30 ml/min, and large increases in plasma BGP did not occur until the 24-h creatinine clearance was below 20 ml/min. We conclude that, in normal subjects and patients with mild to moderate renal failure, plasma elevations of BGP reflect increased bone turnover rather than decreased renal clearance.     

Biological monitoring of aluminium in renal patients

During the past decade, aluminium (Al) has been shown to possess a potential for systemic toxicity. Renal patients form a high-risk group for Al poisoning, and biological monitoring is indicated to diagnose and prevent toxicity. Electrothermal atomic absorption spectrometry is the analytical method of choice for measuring Al levels. Special precautions have to be taken to prevent contamination with environmental Al or adsorption of Al to glassware during analysis. Since this metal is almost evenly distributed between plasma and erythrocytes, either plasma or whole blood may be used to estimate exposure to Al. Measurement in dialysis fluid is suitable to assess parenteral uptake. Hair analysis is of no value. The normal value in plasma is 7.3 +/- 2.0 micrograms/l (mean +/- SD, n = 10); toxic symptoms are mainly associated with levels above 100 micrograms/l. A dialysate level not exceeding 5 micrograms/l may be considered safe; intestinal absorption is then the only remaining, highly variable source of Al uptake.     

An improved specific and sensitive radioenzymatic method for determination of serotonin concentrations in biological fluids (radioenzymatic serotonin method)

Existing radioenzymatic assays for determining the concentration of serotonin in biological fluids have been further modified resulting in increased sensitivity and a lowered detection limit (0.017 pmol). The assay has been used for determinations in platelet-poor plasma (3.3-20.5 nmol/l) and cerebrospinal fluid (0.15-20 nmol/l) from man.     

Vitamin D therapy in clinical practice. One dose does not fit all

INTRODUCTION: Vitamin D is given to most patients with osteoporosis particularly the elderly and those on bisphosphonates. The most widely advocated dose is 800 IU with or without calcium. Whether or not this enables all or most patients to become vitamin D replete in clinical practice is not established. AIMS: This study investigated a large cohort of patients with osteoporosis attending a metabolic bone clinic to identify if those on vitamin D supplements were adequately treated and if those commenced on treatment developed normal vitamin D levels. METHODS: Twenty-five hydroxy vitamin D measurements from new all patients attending a district general hospital metabolic bone clinic as part of their preclinic investigations was examined. It was noted as to whether or not they were taking calcium and or vitamin D supplements. Patients not on supplements but with a low baseline vitamin D were treated with supplements and then had a repeat measurement after at least 3 months to assess whether or not they were replete. RESULTS: From the database of 1028 patients, 100 had preclinic and follow-up vitamin D levels. They were of average age 61 years (SD 12) with a mean baseline vitamin D of 26 nmol/l. The mean posttreatment level was 58 nmol/l (SD 25). Posttreatment vitamin D levels were < 60 nmol/l in 55%, < 50 nmol/l in 36%, < 40 nmol/l in 24% and < 30 nmol/l in 13% and < 20 nmol/l in 4%. In 41 patients on Calcichew D3 Forte two tablets per day pretreatment vitamin D was 24 nmol/l (SD 16) and posttreatment 62 nmol/l (SD 28). Of this subgroup posttreatment 41% were < 60 nmol/l, 27% < 50 nmol/l, 22% < 40 nmol/l and 10% < 30 nmol/l. Two hundred and ten patients on vitamin D treatment preclinic had a mean vitamin D level of 64 nmol/l (SD 28). One hundred and twenty-four patients already on two tablets of Calcichew D3 Forte per day had a mean of 68 nmol/l (SD 28) of whom 38% were < 60 nmol/l, 24% < 50 nmol/l, 16% < 40 nmol/l, 6% < 30 nmol/l and 3% < 20 nmol/l. CONCLUSION: Vitamin D therapy with conventional treatment improves serum levels of 25 hydroxy vitamin D but still leaves some patients with significant insufficiency and therefore the same dose of vitamin D is not appropriate for all.