A simple, rapid assay for plasma oxalate in uraemic patients using oxalate oxidase, which is free from vitamin C interference

An enzymatic assay for the determination of oxalate in plasma was developed which is specific, simple, rapid and requires no specialised equipment; interference from vitamin C was removed by incubation of acidified plasma ultrafiltrate with ascorbate oxidase prior to oxalate estimation. Recoveries were 93 +/- 11% and the inter-batch coefficient of variation for 31 determinations at an oxalate level of 24 mumol/l was 10%. The assay is linear up to 300 mumol/l with a detection limit of 2 mumol/l. The reference range, based on results from 25 healthy volunteers, was defined as less than 2-5 mumol/l which is similar to levels established for the in vivo isotope dilution technique. The assay has an added advantage over the latter method, which requires a urine collection, in that it can be applied to plasma from anuric patients. A linear correlation (r = 0.68, p less than 0.001) was found between plasma oxalate and serum creatinine in individuals with varying degrees of renal failure.     

Plasma glycolate concentrations under conditions of dialysis

Plasma glycollate and oxalate concentrations were measured in 20 patients undergoing chronic haemodialysis treatment. The mean plasma glycollate level was 173.7 +/- 52.9 mumol/l, which was not significantly different from the normal value (means = 145.8 +/- 37.8 mumol/l). The mean plasma oxalate concentration (means = 128.7 +/- 25.6 mumol/l) was about 8 times higher than the value found in normal volunteers (means = 16.8 +/- 6.0 mumol/l). During haemodialysis lasting for 6 hours the plasma oxalate concentration decreased by 53.5%. However, no decline in plasma glycollate levels was noted. Since glycollate was not found in ultrafiltrates obtained in vivo, it is concluded that glycollate is not eliminated during haemodialysis treatment.     

The determination of plasma oxalate concentrations using an enzyme/bioluminescent assay

An enzyme/bioluminescent assay for the determination of oxalate in plasma is described in which NADH, a reaction product of the enzymic degradation of oxalate by oxalate decarboxylase and formate dehydrogenase, is determined using a commercially available bioluminescent system. In contrast to most previously documented methods, this sensitive and specific assay requires minimal sample preparation allowing oxalate concentrations to be determined within 2 h of sample collection. The limit of detection for plasma samples is 0.8 mumol/l. The recovery of oxalate added to plasma averaged 99%. The inter-batch coefficient of variation, calculated by analysis of a plasma sample from a uraemic patient (oxalate concentration = 45.8 mumol/l) on 8 occasions, over a period of 5 wk, was 3.2%. Plasma oxalate concentrations in 35 normal subjects ranged from less than 0.8-1.5 mumol/l, which is in excellent agreement with values obtained by in vivo isotope dilution studies. Plasma oxalate was found to be strikingly elevated in a group of uraemic patients maintained on regular haemodialysis.     

An inexpensive method for sensitive enzymatic determination of oxalate in urine and plasma

In this simple, sensitive, and rapid enzymatic method for the determination of oxalate in urine or plasma, oxalate oxidase (EC 1.2.3.4) prepared from barley seedlings is used to convert oxalate to carbon dioxide and hydrogen peroxide, which is determined photometrically at 600 nm, with use of horseradish peroxidase, by oxidative coupling of 3-methyl-2-benzothiazoline hydrazine with N,N-dimethylaniline. Plasma is pre-treated by ultrafiltration and co-precipitation of oxalate with calcium sulfate and ethanol, urine by dilution and reversed-phase chromatography on C18 columns. Analytical recovery for urine is 99 +/- 2%, for plasma 92 +/- 3%. The normal range for urinary excretion is 0.10 to 0.56 mmol/24 h, and for the concentration in plasma 0.4 to 3.7 mumol/L. There were no significant sex-related differences in urinary excretion or plasma concentration. Our within- and between-assay coefficients of variation were, respectively, less than 3.4% and less than 6.0% for urine, and less than 1.5% and less than 4.3% for plasma.     

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.     

Gas-chromatographic estimation of urinary oxalate and its comparison with a colorimetric method

We describe a simple, specific gas-chromatographic method for urinary oxalate. Its specificity was evaluated by precipitating the oxalate as its calcium salt from urine, followed by methylation of the oxalate with boron trifluoride/methanol and subsequent gas-chromatographic separation and quantitation of the dimethyl oxalate. [U-14C]Oxalate and n-decanoic acid are used as internal standards. Analytical recoveries ranged from 93.8 to 97.7% for oxalate-supplemented urine. Twenty replicate analyses of urines containing typical concentrations of oxalate gave CVs of 10.0, 9.1, and 8.3% for low, medium, and high concentrations, respectively. Day-to-day precision (CV) for single analyses repeated on 20 days was 8.6% for the low urinary oxalate concentration. The lower limit of detection of urinary oxalate is 25 mumol/L. Results by our method correlated well (r = 0.95) with those by a colorimetric method (Clin. Chim. Acta 36: 127, 1972) but averaged 68.4% of those obtained colorimetrically (n = 75 samples, p less than 0.001). The expected range for our method is calculated to be 80 to 500 mumol of oxalate per 24-h urine (mean +/- 1 SD: 280 +/- 100).     

Relaxing effects of cyclic GMP and cyclic AMP-enhancing agents on the long-lasting contraction to endothelin-1 in the porcine coronary artery

In the coronary circulation, endothelin-1 (ET-1) evokes spasms which are difficult to treat when the endothelial integrity is compromised. This study compares several classes of relaxing agents on already established contractions to ET-1 in an in vitro model using ring segments of the porcine left descending coronary artery (pLAD). All segments were precontracted with 10 nmol/L ET-1. The calcium channel blocker isradipine was 300 times more potent than verapamil, but was only a partial relaxant; the maximal relaxation obtained was 52 +/- 2% (n = 6). Atrial natriuretic peptide (ANP) was an equally potent relaxant of the ET-1 contraction; however, it too was an incomplete relaxant, maximal relaxation being < 60%. A 50% relaxation of the ET-1 contraction was obtained with 0.28 +/- 0.24 mumol/L ANP, n = 4 (IC50). Comparison of cyclic nucleotide analogues revealed a 30 times higher potency for 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP)(IC50 44 +/- 11 mumol/L, n = 6) than for 8-bromo-cyclic adenosine monophosphate (8-Bi-cAMP) (IC50 1600 mumol/L, n = 6). The cyclic nucleotide phosphodiesterase (PDE) inhibitor milrinone, a PDE 3-inhibitor with an IC50 2.4 +/- 1.8 mumol/L, (n = 6) was 10 times more potent than rolipram (PDE 4-inhibitor), zaprinast (PDE 5-inhibitor) and vinpocentine (PDE 1-inhibitor). Withdrawal of these analogues and inhibitors from segments continuously exposed to 10 nmol/l ET-1 revealed that vinpocentine and 8-Br-cGMP were irreversible relaxants, in contrast to milrinone and 8-Br-cAMP. In conclusion, this study has demonstrated that cGMP-enhancing agents, such as the naturally occurring ANP, the calcium channel blocker isradipine, and the synthetic inhibitor of PDE 3, were the most effective relaxants of ET-1 evoked contractions in pLAD in vitro.     

Allantoin as a marker of oxidative stress in human erythrocytes

Abstract Background: Uric acid is the final product of purine metabolism in humans. It was determined that this compound has important antioxidative properties and it may be oxidized to allantoin by various reactive oxygen species. Therefore, the measurement of allantoin may be useful for the determination of oxidative stress in humans. Methods: We measured allantoin and uric acid in human plasma and erythrocytes obtained from patients with chronic renal failure before hemodialysis (n=30) and blood donors (n=30). We used a method based on selective isolation of allantoin from deproteinized plasma and erythrocyte lysate samples on AG 1-X8 resin and its derivatization to glyoxylate-2, 4-dinitrophenylhydrazone. Separation of glyoxylate-2, 4-dinitrophenylhydrazone from interfering substances was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. Uric acid was determined by reversed phase HPLC with UV/VIS detection at 292 nm. Results: We found significant differences in allantoin and uric acid concentration between the patients with chronic renal failure and the control group both in plasma (20.5+/-6.5 mumol/L and 323.9+/-62.9 mumol/L vs. 2.1+/-1.1 mumol/L and 270.1+/-62.3 mumol/L, p<0.05) and erythrocytes [82.8+/-39.1 nmol/g hemoglobin (Hb) and 110.7+/-28.8 nmol/g Hb vs. 20.1+/-6.1 nmol/g Hb and 82.1+/-23.7 nmol/g Hb, p<0.05]. Conclusions: Significant higher levels of allantoin in both plasma and erythrocytes of patients with chronic renal failure indicate that allantoin may be used as a good marker of oxidative stress. Clin Chem Lab Med 2008;46:1270-4.     

Evidence that serum calcium oxalate supersaturation is a consequence of oxalate retention in patients with chronic renal failure.

Serum oxalate rises in uremia because of decreased renal clearance, and crystals of calcium oxalate occur in the tissues of uremic patients. Crystal formation suggests that either uremic serum is supersaturated with calcium oxalate, or local oxalate production or accumulation causes regional supersaturation. To test the first alternative, we ultrafiltered uremic serum and measured supersaturation with two different methods previously used to study supersaturation in urine. First, the relative saturation ratio (RSR), the ratio of the dissolved calcium oxalate complex to the thermodynamic calcium oxalate solubility product, was estimated for 11 uremic (before and after dialysis) and 4 normal serum samples using a computer program. Mean ultrafiltrate oxalate predialysis was 89 +/- 8 microM/liter (+/- SEM), 31 +/- 4 postdialysis, and 10 +/- 3 in normals. Mean RSR was 1.7 +/- 0.1 (predialysis), 0.7 +/- 0.1 (postdialysis), and 0.2 +/- 0.1 (normal), where values greater than 1 denote supersaturation, less than 1, undersaturation. Second, the concentration product ratio (CPR), the ratio of the measured calcium oxalate concentration product before to that after incubation of the sample with calcium oxalate monohydrate crystal, was measured in seven uremic and seven normal serum ultrafiltrates. Mean oxalate was 91 +/- 11 (uremic) and 8 +/- 3 (normal). Mean CPR was 1.4 +/- 0.2 (uremic) and 0.2 +/- 0.1 (normal). Predialysis, 17 of 18 uremic ultrafiltrates were supersaturated with respect to calcium oxalate. The degree of supersaturation was correlated with ultrafiltrate oxalate (RSR, r = 0.99, r = 29, P less than 0.001; CPR, r = 0.75, n = 11, P less than 0.001). A value of ultrafiltrate oxalate of 50 microM/liter separated undersaturated from supersaturated samples and occurred at a creatinine of approximately 9.0 mg/dl.     

The determination of plasma oxalate concentrations using an enzyme/bioluminescent assay. 2. Co-immobilisation of bioluminescent enzymes and studies of in vitro oxalogenesis

An inexpensive, continuous flow assay for the determination of oxalate in plasma is described. The assay is based on the bioluminescent determination of NADH, a product of the degradation of oxalate by oxalate decarboxylase and formate dehydrogenase, using bioluminescent enzymes immobilized on cyanogen bromide-activated sepharose. The detection limit of the assay is 0.8 mumol/l. Intra-batch CV values of 5.2 and 3.8% were obtained at oxalate concentrations of 18 and 60 mumol/l. Recovery of added oxalate averaged 100.7%. Plasma oxalate ranged from less than 0.8 to 2 mumol/l in 14 healthy subjects, and from 6 to 134 mumol/l in 125 patients with renal disease treated by continuous ambulatory peritoneal dialysis. Ascorbic and dehydroascorbic acid did not directly interfere in the assay. In vitro oxalogenesis was observed in blood from 12 healthy subjects, but only after samples had stood at room temperature for more than 6 h. No significant oxalate generation occurred in blood from 24 patients with impaired renal function, even after standing at room temperature for 24 h. Oxalate generation was inhibited by the addition of oxalate to plasma, but the addition of urea and creatinine was without effect.     

Determination of protein-bound urinary gamma-carboxyglutamic acid in calcium nephrolithiasis.

Nephrocalcin is a urinary gamma-carboxyglutamic acid (Gla) containing protein that may be a physiological inhibitor of calcium oxalate nephrolithiasis. Nephrocalcin isolated from urine of stone formers seems to be abnormal in lacking Gla that is required for inhibitory activity. In order to study this hypothesis, we compared the protein-bound urinary Gla contents in 32 calcium oxalate stone formers and in 24 controls. Protein-bound Gla was resolved by reversed-phase high-performance liquid chromatography after elimination of free Gla, alkaline hydrolysis and precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Protein-bound urinary Gla concentrations were similar in stone formers (0.83 +/- 0.38 mumol/l, mean +/- SD) and controls (0.81 +/- 0.27) and were less than 5% of free urinary Gla. However, excretion rates of free and protein-bound Gla (nmol/min) were higher in stone formers (P = 0.006 and P = 0.002). Positive correlations (P = 0.000) were observed between free and protein-bound Gla both in controls and in stone formers. These results do not support the hypothesis of a lacking Gla nephrocalcin in stone formers.     

The quantitation of oxalate in amniotic fluid by ion-chromatography

Calcium oxalate is the predominant constituent of most kidney stones. The rare genetic disorder, primary hyperoxaluria, is characterized by the continuous excessive synthesis and urinary excretion of oxalic acid, leading to stone formation and renal insufficiency. The earliest measurement of oxalate in suspected cases of primary hyperoxaluria is advantageous and would lend support for continued analysis and eventual confirmation of the disease. Therefore, we quantitated oxalate levels in amniotic fluid (AF) using medium pressure ion-exchange chromatography. The mean concentration of oxalate in amniotic fluid was 1.67 mg/l +/- 0.8 (SD); (range 0.64 to 5.11 mg/l). The mean oxalate/creatinine ratio (O/C) was 0.23 +/- 0.11 (SD); (range 0.07 to 0.53). This ratio is similar to that found in the urine of infants less than 1 year (0.19 +/- 0.10; n = 17). There was no significant difference between males and females in oxalate concentration or O/C ratio. Regression analysis showed no significant correlation of fetal age with oxalate, O/C or creatinine. Studies in 13 sets of di-amnionic twins showed no statistical difference in oxalate or O/C between twin A and B. This study demonstrates the ability to accurately quantitate oxalate in amniotic fluid by ion-chromatography, and suggests that this may have a potential application in the initial screening process for the prenatal detection of primary hyperoxaluria.     

Modified enzyme-based colorimetric assay of urinary and plasma oxalate with improved sensitivity and no ascorbate interference: reference values and sample handling procedures.

The measurement of oxalate in urine and plasma continues to be difficult, particularly in the presence of ascorbate. We have modified and validated a colorimetric assay involving the use of oxalate oxidase (EC 1.2.3.4). Modification of an HPLC spectrophotometric detector improved sensitivity (to as much as 1000-fold that of conventional spectrophotometers) and allowed measurement of oxalate concentrations less than 1 mumol/L. This provided more than enough sensitivity for measurement of normal concentrations of plasma oxalate. We established reference values for oxalate concentrations in urine and plasma, studied sample handling, and established conditions to avoid ascorbate interference in urine and plasma measurements. Mean analytical recovery of [14C]oxalate from plasma to the filtrate was 86 (SD 10)%; recovery of unlabeled oxalate from filtrate was 87 (SD 9)%. Urinary oxalate excretion rates in apparently healthy controls were 0.11-0.46 mmol/24 h. Plasma concentrations in control subjects were 2.5 (SD 0.7) mumol/L, similar to concentrations determined by recent gas chromatographic and isotope dilution methods. Frozen and acidified urine samples showed no interference from ascorbate when excess ascorbate was avoided. Ingestion of 2 g of ascorbate daily did not increase urinary oxalate in healthy control subjects, but during storage ascorbate was converted to oxalate in all conditions tested.     

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.     

Serum calcium oxalate saturation in patients on maintenance haemodialysis for primary hyperoxaluria or oxalosis-unrelated renal diseases.

1. The serum oxalate concentration rises in chronic renal failure and it is only partially eliminated by regular dialysis treatment. However, the recent literature is not conclusive on whether progressive oxalate retention and secondary oxalosis should be expected in patients on regular dialysis treatment. 2. To further investigate this, we have estimated the state of saturation with respect to calcium oxalate monohydrate in plasma ultrafiltrates from 28 patients on maintenance haemodialysis and eight healthy control subjects, matched for sex and age. Five patients had type I primary hyperoxaluria and histologically proven oxalosis, whereas 23 had oxalosis-unrelated renal diseases. Dialysis efficiency was quantified as the KdTd/V of urea. Samples were obtained from each patient before, immediately after and 48 h after a dialysis session. Fasting samples were obtained from the control subjects. Oxalate was determined in both plasma ultrafiltrates and the whole dialysate by ion-exchange chromatography, after a non-delayed and [14C]oxalate-recovery-controlled procedure. The state of saturation with calcium oxalate monohydrate was estimated by means of a computer system which solved the interactions among 45 complex species. 3. The fasting plasma oxalate concentration (means +/- SD) in ultrafiltrates from healthy subjects was 3.8 +/- 1.5 (range 1.4-5.8) mumol/l, and the state of saturation with calcium oxalate monohydrate was 0.096 +/- 0.04.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioimmunoassay of human serum serotonin

A radioimmunoassay for extracted, N-acetylated human serum serotonin (5-hydroxytryptamine) is described. Antisera were raised in rabbits against a conjugate of bovine serum albumin with serotonin hemisuccinamide. Polyethylene glycol in combination with anti-rabbit immunoglobulins was used to separate bound and unbound 125I-Bolton Hunter-serotonin conjugate. Ethanol precipitation of serum proteins was used to extract serotonin, which was subsequently acetylated with acetic anhydride to N-acetyl serotonin. The average recovery was 66%. The minimal detectable concentration of N-acetyl serotonin was 0.012 mumol/l serum (25 fmol per tube). The intra-assay precision (CV) was 6.8% (n = 20) at a level of 0.9 +/- 0.06 mumol/l. The inter-assay CV was 10% at a level of 0.49 +/- 0.049 mumol/l, and 25% (n = 10) at a level of 2.16 +/- 0.53. Analytical recovery of serotonin, corrected for losses during extraction and acetylation, was 99 +/- 13%. The only substance cross-reacting with the antibody was endogenous N-acetyl serotonin. This was detectable when the acetylation step was omitted, and it can be removed by extraction before the acetylation. The observed range for the concentration of serotonin in serum was for 59 women 0.45 - 3.46 (mean +/- SD: 1.37 +/- 0.63 mumol/l) and for 59 men 0.19 - 2.8 (mean +/- SD: 1.18 +/- 0.56 mumol/l). All values are corrected for endogenous N-acetyl serotonin: observed range 0 - 0.18 (mean +/- SD: 0.03 +/- 0.03 mumol/l).     

The determination of oxalate in haemodialysate and plasma: a means to detect and study 'hyperoxaluria' in haemodialysed patients

In order to find out whether hyperoxaluria can be demonstrated in patients on chronic (twice a week) haemodialysis, a group of 13 patients was investigated. These included one patient with proven primary hyperoxaluria, one suspected of having this disease and 11 patients in whom no information was available as to their oxalate metabolism. Oxalate concentrations in haemodialysate fractions and blood samples, taken before and after dialysis, were determined. The patient with primary hyperoxaluria had a plasma oxalate concentration before dialysis above 100 mumol/l and after dialysis above 25 mumol/l, while the oxalate concentration in haemodialysate at the start of dialysis was above 25 mumol/l and at the end above 10 mumol/l. The patient suspected of hyperoxaluria had similar values. Of the remaining 11 patients, one was shown to exhibit a transient hyperoxaluria, but the others showed a normal oxalate metabolism. A plasma oxalate/creatinine concentration ratio exceeding 0.1, and the calculated total quantity of oxalate removed by dialysis exceeding 2 mmol, also enabled a diagnosis of hyperoxaluria to be made. Hyperoxaluria can still be demonstrated in patients, who because of renal failure are subjected to haemodialysis. Measurements of oxalate in haemodialysate and plasma are valuable in cases where kidney transplantations are considered, especially when the particular patient exhibits hyperoxaluria.     

Oxalate measurement in the picomol range by ion chromatography: values in fasting plasma and urine of controls and patients with idiopathic calcium urolithiasis

Oxalate was measured by ion chromatography in the ultrafiltrate of heparinized plasma from peripheral venous blood, using a membrane with a cut-off molecular weight (Mr). The following criteria were established: sensitivity 0.7 mumol.l-1; intra- and inter-assay coefficients of variation 4% and 12%, respectively; precision of duplicate determinations (expressed as standard deviation) 0.08 mumol.l-1; overall recovery (oxalate added and diluted, respectively) 100.7%. These qualified the method for assessment of plasma oxalate in healthy human controls (males: n = 12) as well as patients with idiopathic renal calcium urolithiasis (males: n = 22; females: n = 16). Renal calcium urolithiasis patients were subclassified into those with normocalciuria and idiopathic hypercalciuria. In male and female controls the mean values (and range) of plasma oxalate were 1.98 (1.4-2.5) and 1.78 (0.7-2.9) mumol.l-1, respectively. In male controls ultrafiltration (membrane cut off Mr 10,000) revealed that 11-16% plasma oxalate was bound to constituents having an apparent Mr above 10,000, and that with use of membranes with smaller pore size, the ultrafilterability of oxalate decreases further. In renal calcium urolithiasis the following values were elicited (mumol.l-1): male normocalciuria 1.78 (0.8-4.0), idiopathic hypercalciuria 1.58 (1.2-2.2); female normocalciuria 1.69 (0.8-3.6), idiopathic hypercalciuria 1.21 (0.8-2.1). The difference from controls is significant in idiopathic hypercalciuria (males and females). In contrast, in fasting urine of renal calcium urolithiasis the oxalate excretion rate (5-45 mumol per 120 min) and oxalate clearance (21-328 ml per min) resemble those in controls, whereas in renal calcium urolithiasis the fractional oxalate clearance (30-357% of creatinine clearance) tended to higher values (p less than 0.01, in male idiopathic hypercalciuria versus controls). It is suggested that 1) ion chromatography allows the reliable assessment of ultrafiltrable plasma oxalate in health and disease states, 2) in renal calcium urolithiasis this technique may help to elucidate oxalate pathophysiology, especially the mode of renal handling of oxalate.     

High-performance liquid chromatographic determination of plasma glycolic acid in healthy subjects and in cases of hyperoxaluria syndromes

A liquid chromatographic procedure for the determination of glycolic acid in plasma is proposed. The system is based on pre-column derivatization of the alpha-keto acid by means of phenylhydrazine, coupled with the enzymatic oxidation of glycolate to glyoxylate. The phenylhydrazone formed is separated by reversed-phase liquid chromatography and detected by UV absorption. The measured within and between-batch CV imprecision was 2.6 and 11.3%, respectively, at 5.68 mumol/l glycolate concentration; the analytical recovery was 102.0 +/- 7.3% and the minimum detectable concentration of glycolate was 0.3 mumol/l. The reference interval for plasma glycolate was 4.51 to 12.20 mumol/l (n = 14). Results of determinations of plasma samples from uremic patients, patients with type I primary hyperoxaluria and patients with chronic renal failure secondary to systemic oxalosis are reported.     

Free choline in human plasma analysed by simple radio-enzymatic procedure: age distribution and effect of a meal

Ultrafiltration of plasma was shown to be a simple and rapid method to obtain a stable sample for direct measurement of free choline (Ch) in plasma by a radioenzymatic procedure. Free Ch was analysed in plasma from healthy volunteers fasted 12-15 h and 1 h after a meal. The free Ch concentration was found within narrow limits with a mean of 10.6 +/- 0.4 mumol/l in the fasted subjects and 11.5 +/- 0.3 mumol/l 1 h after a meal. The difference is significant (paired t test, P less than 0.01, n = 23). Dietary influence on the free Ch concentration in human plasma is suggested. In three newborn infants (1-3 min post partum) the Ch concentration in plasma from the umbilical vein was 24.5 +/- 1.9 mumol/l.