Hypoxanthine-guanine phosphoribosyl transferase: assay using high performance liquid chromatography

A method is described for the estimation of hypoxanthine-guanine phosphoribosyltransferase using high performance liquid chromatography.The inosine monophosphate (IMP) generated from hypoxanthine is determined, after separation on a C₁₈ reversed phase silica column with a buffer-methanol gradient, by the absorbance at 254 nm. Simultaneous reciprocal measurement of hypoxanthine consumption is made. The assay is suitable for screening red cell lysates for hypoxanthine-guanine phosphoribosyltransferase deficiency; the results being expressed as nmol inosine monophosphate · h^?1 · mg^?1 haemoglobin.The normal range found was 94 ± 15 nmol IMP-h^?1 · mg^?1 haemoglobin and hypoxanthine-guanine phosphoribosyltransferase activities down to 1% of normal can be assayed accurately.     

A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A)

4-Methylumbelliferyl-beta-D-galactopyranoside-6-sulphate was synthesized and used for the determination of galactose-6-sulphate sulphatase activity. Fibroblasts and leucocytes from 12 different Morquio A patients, showed 0.0-2.7% of mean normal galactose-6-sulphate sulphatase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-galactopyranoside-6-sulphate requires the sequential action of galactose-6-sulphate sulphatase and beta-galactosidase. Normal beta-galactosidase activity caused nearly complete hydrolysis of non-fluorescing 4-methylumbelliferyl-galactoside, formed during incubation. In cell extracts with a beta-galactosidase deficiency however, a second incubation in the presence of excess beta-galactosidase is needed to avoid underestimation of galactose-6-sulphate sulphatase activity.     

A rapid and simple radiometric assay for thymidine phosphorylase of human peripheral blood cells

A radiometric method is described for measuring thymidine phosphorylase activity in human peripheral blood cells. The substrates [¹⁴C] thymidine or [¹⁴C]thymine are converted to the base or deoxynucleoside, respectively, and two alternative Chromatographic methods to isolate the products of the reaction have been employed. With the described methods the specific activity for thymidine phosphorylase in human lymphocytes is 0.21 ± 0.08; monocytes 0.21 ± 0.16 and granulocytes 0.17 ± 0.02 μmol δ h^?1 δ mg^?1 protein. For human T- or null lymphoblasts, thymidine phosphorylase activity was found to be approximately 10% of that of B lymphoblasts.     

Tandem mass spectrometry for the direct assay of enzymes in dried blood spots: application to newborn screening for mucopolysaccharidosis II (Hunter disease)

BACKGROUND: A treatment for mucopolysaccharidosis II (Hunter syndrome) has recently become available. Therefore, we developed a high-throughput assay method appropriate for newborn screening for the relevant enzyme, iduronate 2-sulfatase. METHODS: We synthesized a new iduronate 2-sulfatase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard. The assay uses a dried blood spot on a newborn screening card as the enzyme source. RESULTS: When the assay was tested on dried blood spots, the iduronate 2-sulfatase activity measured for 13 patients with Hunter syndrome was well below the interval found for 57 randomly chosen newborns. The assay was more sensitive than previously reported iduronate 2-sulfatase assays. CONCLUSIONS: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of Hunter syndrome. This method also has the potential to be carried out in multiplex fashion to assay several different enzymes relevant to lysosomal storage diseases that are assayed in a single infusion into the mass spectrometer.     

Differences in phospholipase A2 activity between males and females and Asian Indians and Caucasians

There is epidemiological evidence that chronic inflammatory diseases occur more frequently in female than in male subjects and prevail differently in various ethnic populations. Phospholipase A₂ (PLA₂) (group II) plays a key role in many inflammatory reactions by releasing free arachidonic acid, which is a prerequisite for the production of proinflammatory lipid mediators. We therefore, measured PLA₂ activity in plasma, serum, leucocytes and lymphocytes in 20 female and 20 male subjects, 10 of each group being of Asian Indian and of Caucasian origin respectively. When PLA₂ activity was measured in crude plasma and serum no dependency from gender and ethnicity was observed. Following acid extraction and heating, PLA₂ activity in plasma was higher in Caucasians (27.8 ± 2.2 nmol L^?1 mg ^?1 protein 60 min^?1) than in Asian Indians (17.9 ± 2.5 nmol L^?1 mg^?1 protein 60 min^?1) (P < 0.005) and higher in females (28.5 ± 2.6 nmol L^?1 mg^?1 protein 60 min^?1) than in males (17.3 ± 2.0 nmol L^?1 mg^?1 protein 60 min^?1) (P < 0.001). Similar differences were observed when only Asian Indian or Caucasian females were compared with their corresponding males. Contrary to plasma, in which the specific activity of PLA₂ increased following acid extraction and heating, the activity was completely abrogated in serum after extraction and heating. Lymphocytes exhibited lower activities of PLA₂ than neutrophils in all four groups of subjects investigated. Females had a tendency towards higher PLA₂ activity in both lymphocytes and neutrophils than males. In conclusion the present investigation revealed an ethnic- and sex-dependent basal activity of PLA₂, a key enzyme in the pathogenesis of chronic inflammatory diseases.     

An improved method for the assay of platelet pyruvate dehydrogenase

An improved method for the assay of human platelet pyruvate dehydrogenase is described. By generating the substrate [1-¹⁴C]pyruvate in situ from [1-¹⁴C]lactate plus l-lactate dehydrogenase, the rate of spontaneous decarboxylation is dramatically reduced, allowing far greater sensitivity in the assay of low activities of pyruvate dehydrogenase. In addition, no special precautions are required for the storage and use of [1-¹⁴C]lactate, in contrast to those for [1-¹⁴C] pyruvate. These factors allow a 5–10-fold increase in sensitivity compared with current methods. The pyruvate dehydrogenase activity of normal subjects as determined by the [1-¹⁴C]lactate system was 215 ± 55 pmol · min^?1 · mg^?1 protein (n = 18). The advantages of this assay system are discussed.     

Prostate specific acid phosphatase: further studies with immunological techniques

Prostatic acid phosphatase was purified from prostatic adenomas. The procedure involved chromatography on Concanavalin A-Sepharose, DEAE-cellulose, Bio-Gel P-150 and L-tartrate-Sepharose. The purified phosphatase hydrolyzed p-nitrophenyl phosphate at a rate of 270 μmol · mg^?1 min^?1 (25°C) and showed homogeneity upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The final prostatic acid phosphatase preparation was pure and the antisera were monospecific as judged by the highly sensitive technique of crossed immunoelectrophoresis. Of the procedures evaluated for the iodination of the purified enzyme, oxidation with lodogen was found to give the best iodinated product.     

Effects of di-isopropylfluorophosphate and isopropanol on the measurement of plasma renin activity

Since the protease inhibitor, di-isopropylfluorophosphate (DFP), inhibits the cryoactivation of inactive renin, its addition to stored plasma should prevent unintended renin activation and thereby minimize this source of inaccuracy in the assay of plasma renin activity. However, we have found that 6 mmol/1 DFP in stored frozen plasma actually reduced plasma renin activity from 4.89 ± 0.13 (S.D.) to 2.48 ± 0.17 ng · ml^?1 · h^?1 (p < 0.001). This effect did not occur if DFP was added immediately before assay, suggesting that the decreased renin activity reflected an action of DFP on components of the renin system during storage rather than any interference with the assay method itself. The organic solvent isopropanol, which is required to dilute the DFP, appeared responsible for this phenomenon, for when the isopropanol alone, in concentrations of 20 and 40 μl/ml, was added to stored frozen plasma it decreased plasma renin activity from 3.6 ± 0.3 to 2.2 ± 0.1 and 0.6 ± 0.1 ng · ml^?1 · h^?1, respectively (p < 0.001 in each case). Correspondingly, plasma renin substrate concentrations were decreased to 71% and 6% of control, indicating that the renin activity reductions produced by isopropanol were due to its denaturation of substrate. Moreover, addition of exogenous sheep renin substrate (1400 ng/ml) immediately before assay restored plasma renin activity to control. Thus, although DFP effectively prevents inadvertent renin activation in stored frozen plasma, it would seem important that subsequent assays for plasma renin activity be performed in the presence of added exogenous substrate.     

Dosage de l'activité adénosine désaminasique dans les érythrocytes et les lymphocytes humains

A radiochromatographic method is described for measuring enzymatic activity of adenosine deaminase in human erythrocytes and lymphocytes. [8-¹⁴C]-adenosine is converted into inosine and hypoxanthine; after Chromatographie separation of the products, the radioactivity is determined.The kinetic properties of the enzyme have been studied. The K_m values for the erythrocyte and lymphocyte enzymes are higher as compared with purified deaminase. Optimum conditions for substrate concentration for assay were established.The mean normal activity (± S.E. of mean) is: for erythrocytes, 494 ± 61; nmol min^-1 ml^-1; for lymphocytes, 147 ± 0.18 nmol min^?1 10⁶ cellules.The mean values are higher than that given by other methods working at a lower (non-staurating) substrate concentration.     

Increased serum angiotensin converting enzyme activity in type T insulin—dependent diabetes mellitus: its relation to metabolic control and diabetic complications

Abstract. Serum angiotensin-converting enzyme (ACE) was measured in 150 insulin-dependent diabetes mellitus (IDDM) patients and 72 healthy subjects by radioassay, using [³H]-hippuryl-glycyl-glycine as a substrate. Mean (SD) serum ACE activity in diabetic patients was 120 ± 33 nmol ml^?1 min^?1 (range 46–215) and was significantly increased by 56% compared to control values (77 ± 23 nmol ml^?1 min^?1, range 46–125, P < 0·001). ACE activity > 125 nmol ml^?1 min^?1 was observed in 60 of 150 IDDM patients. 96 IDDM patients were normoalbuminuric (< 22 mg 24 h^?1) and 49 patients were micro- or macroalbuminuric (range 22–6010 mg 24 h^?1). Micro- and macroalbuminuric IDDM patients were found to have significantly greater ACE activity values than normoalbuminuric patients (128 ± 36 vs. 115 ± 30 nmol ml^?1 min^?1, P = 0·025). Metabolically well-controlled IDDM patients (glycosylated haemoglobin ≤ 8%) had lower ACE activity values than the patients with glycosylated haemoglobin greater than 8% (109 ± 20 vs. 127 ± 32 nmol ml^?1 min^?1, P < 0·02). A significant correlation between degree of metabolic control and ACE activity was found (r = 0.435, P < 0·001) so that an increase in one glycosylated quartile unit is accompanied by an increase in ACE activity of 10·5 nmol ml^?1 min^?1. Thus ACE activity in the serum of IDDM patients was increased by 56% in 40% of the patients. It was increased in IDDM patients without complications and in patients with retinopathy or nephropathy. In diabetic patients with nephropathy, ACE activity was greater than in diabetic patients without nephropathy. ACE activity was positively correlated with metabolic control. The role of increased ACE activity in the development of diabetic nephropathy remains to be established.     

Phenolsulphotransferase in human tissue: Radiochemical enzymatic assay and biochemical properties

Phenolsulphotransferase (EC 2.8.2.1) (PST) is an important catecholamine and drug metabolizing enzyme. Optimal conditions have been determined for the accurate measurement of PST activity in the human platelet, human renal cortex, and human jejunum with a radiochemical microassay. 3-Methoxy-4-hydroxyphenylglycol (MHPG) and ³⁵S?3'-phosphoadenosine-5'-phosphosulfate (³⁵S-PAPS) were the substrates for the reaction. The apparent Michaelis-Menten (K_m) values for MHPG with platelet, renal cortex, and jejunum were 1.09, 0.46 and 1.16 mmol/l, respectively. Apparent K_m values for PAPS in the same tissues were 0.14, 0.13 and 0.21 μmol/l. The pH optimum of the reaction in all three tissues was approximately 6.2-6.8 with three different buffer systems. The coefficients of variation for the assay of platelet, renal cortex, and jejunal activities were 6.2%, 3.4% and 4.4%, respectively. Mean platelet PST activity in blood samples from 75 randomly selected adult subjects was 5.0 ± 1.72 nmol of MHPG sulfate formed per hour per mg of platelet protein (8.3 × 10^?5 ± 2.9 × 10^?5μmol · min^?1 · mg^?1, mean ± S.D.). There was a 5-fold intersubject variation in platelet PST activity within two standard deviations of the mean value. Experiments in which partially purified human erythrocyte PST was added to platelet, kidney and gut homogenates under these assay conditions provided evidence that endogenous PST inhibitors did not affect the observed enzyme activity.     

Plasma lecithin:cholesterol acyltransferase — reference values and effects of xenobiotics

Plasma lecithin:cholesterol acyltransferase (LCAT) has been measured by an enzymatic method. We did not observe any significant sex variations, but age variations were found. In females, LCAT activities are stable up to 40 years (60 μmol · l^?1 · h^?1 at the 50th centile). Also, from 50 years the median increased progressively to 76 μmol · l^?1 · h^?1. In males, the activity increased from 52 to 71 μmol · l^?1 · h^?1 at the 50th centile in two age groups (15–20 years and 50 years). The effect of some xenobiotics on LCAT activity was studied. We observed an increase in activity of 33% in males when the daily alcoholic beverage consumption ranged from 0 to more than 0.5 litre of wine or beer. LCAT activity increased in children who were treated with hypolipidemic drugs (fenofibrate, Lipanthyl^®). In boys, the mean enzyme activity increased to 35% (p < 0.05). The increase was greater in girls (75%, p < 0.01). Treatment with anticonvulsant drugs gave a decrease in LCAT activity of 32–46%.     

Determination of serum triglycerides by mass fragmentography

A mass fragmentographic reference method for determination of serum triglycerides is described. A fixed amount of a mixture of [1,1,2,2,3,3-²H₅]glycerol tripalmitate and [1,1,2,2,3,3-²H₅]glycerol trioleate (500 nmol) is added to a fixed amount of serum (250 μ1) and extracted with Chloroform/methanol (2:1, v/v). The triglycerides are isolated by means of thin-layer chromatography. The glycerol obtained after acid hydrolysis is converted into the tri-trimethylsilyl derivative and the amount of unlabeled glycerol is determined from the ratio between the recordings at m/e 218 and m/e 222, obtained after analysis with a gas chromatograph-mass spectrometer equipped with an MID (multiple ion detector). The two ions correspond to the peak at M?90 and M?91 in the mass spectrum of the tri-trimethylsilyl derivative of unlabeled and (1,1,2,3,3-²H₅)-labeled glycerol. The relative standard deviation of the method in the range 0.4–5.8 mm 01/1 was 2.4%. A fully enzymatic routine method for determination of triglycerides was compared with the mass fragmentographic reference method. There was a good correlation between the two methods (r = 0.995) and the regression coefficient was 1.19.     

High-affinity binding of folate to a protein in serum of male subjects

The binding pattern of [³H]folate in the serum of male subjects was studied in equilibrium dialysis experiments (pH 7.4, 37°C). It was possible to distinguish between a high (association constant, K_ass = 10¹⁰ (mol/l)^?1; maximum binding of folate = 0.6 nmol/l) and a low affinity type of binding. The highaffinity binder was a trace protein (molecular size ~ 35 000) which was eluted with 30 mmol/l NaCl (pH 6.3) from serum subjected to DEAE-Sepharose ^© CL-6B chromatography. The low-affinity binding activity mainly associated with albumin was eluted with 1 mmol/l NaCl.High-affinity binding was depressed at 7°C (K_ass = 10⁹ (mol/l)^?1). Furthermore, methotrexate acted as a weak inhibitor of high-affinity binding, the molar methotrexate/folate ratio being $\text{100}\text{1}$ at 50% inhibition.     

Increased lipolytic activity of sera from pre-eclamptic women due to the presence of a lysophospholipase

Endresen MJ, Lorentzen B, Henriksen T. Increased lipolytic activity of sera from pre-eclamptic women due to the presence of a lysophospholipase. Scand J Clin Lab Invest 1993; 53: 733-739.

Sera from pre-eclamptic women exhibit an increased lipolytic activity compared to sera of women with normal pregnancies. The null hypothesis of this study was that the increased release of free fatty acids (FFA) was due to hydrolysis of circulating triglycerides. The nature of the increased lipolytic activity was investigated by incubating sera from pre-eclamptic (PE) and normal pregnant women (C) with various lipid substrates radiolabeled in the FFA position.

The release of FFA in PE-sera was not due to hydrolysis of triglycerides or diglycerides. Lysophosphatidylcholine, however, served as substrate for the enhanced lipolytic activity. By using lysophosphatidylcholine with radiolabeled FFA in the sn-1-position we found that 32±10nmol FFAml^_1h^_1 was released in PE-sera, compared to 10±4nmol FFAmP'h^?1 in C-sera. This lysophospholipase activity appears independent of Ca²⁺ and other divalent cations. The increased release of FFA in sera of pre-eclamptic women can be explained by the presence of a lysophospholipase which releases the remaining fatty acid of lysophosphatidylcholine.     

Development of a HPLC-FL method to determine benzaldehyde after derivatization with N-acetylhydrazine acridone and its application for determination of semicarbazide-sensitive amine oxidase activity in human serum

A novel fluorescence labeling reagent N-acetylhydrazine acridone (AHAD) was designed and synthesized. A highly sensitive high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine benzaldehyde after derivatization with AHAD was developed. Optimum derivatization was obtained at 40 °C for 30 min with trichloroacetic acid as catalyst. Benzaldehyde derivative was separated on a reversed-phase SB-C18 column in conjunction with a gradient elution and detected by fluorescence detection at excitation and emission wavelengths of 371 nm and 421 nm. The established method exhibited excellent linearity over the injected amount of benzaldehyde of 0.003 to 5 nmol mL⁻¹. The method was successfully applied to the determination of serum semicarbazide-sensitive amine oxidase (SSAO) activity in humans. SSAO is a significant biomarker because serum SSAO activity is elevated in patients with Alzheimer''s disease, vascular disorders, heart disease and diabetes mellitus. It was demonstrated that the SSAO activity of the hyperglycemic group (60 ± 4 nmol mL⁻¹ h⁻¹) was significantly higher than that of normal blood sugar group (44 ± 4 nmol mL⁻¹ h⁻¹) with P < 0.05.

A highly sensitive HPLC-FL method to determine semicarbazide-sensitive amine oxidase activity was developed utilizing AHAD as the novel fluorescence labeling reagent.     

Day-to-day variation in oxygen consumption and energy expenditure during submaximal treadmill walking in female adolescents

The day-to-day variation in oxygen consumption (V˙O ₂) and energy expenditure (EE) during horizontal treadmill walking was measured using indirect calorimetry in 20 female adolescents (mean age 17·3 years). Two different walking speeds were used: 5 km h^?1 and an individually convenient speed of 3·0 km h^?1 (mean). The two sets of measurements were performed on 2 consecutive days, and great care was taken to minimize possible disturbing factors. The mean V˙O ₂ was 919 ml min^?1 at 5 km h^?1 and 622 ml min^?1 at the individual speed, and the mean values of EE were 4·5 kcal min^?1 and 3·1 kcal min^?1 respectively. The individual day-to-day variation in V˙O ₂ (at 5 km h^?1) was between ?11·7% and +12·6% of the mean V˙O ₂. The coefficient of variation (CV) was 6·4% when values were calculated in ml min^?1 kg^?1. The energy expenditure varied somewhat less between the 2 days (CV = 5·7%). The corresponding value for EE when walking at the individual speed was 7·2%, and the mean day-to day variation in V˙O ₂ was 7·5% (CV). The rate of perceived exertion according to Borg's scale was lower on day 2 (11·9) compared with day 1 (13·0) when walking at 5 km h^?1. There was no difference in heart rate between the 2 days. It is concluded that EE varies somewhat less than V˙O ₂ on successive days, probably because of an interchangeable relationship between breathing gases, depending on which substrate is used for combustion. When using V˙O ₂ and EE for evaluation of physical capacity, the day-to-day variation in the measurements must be taken into consideration.     

A novel mucin sulphatase from human faeces: its identification, purification and characterization.

1. Colonic mucus is heavily sulphated and it is likely that this contributes considerably to its resistance to degradation by bacterial enzymes. The presence of a mucin-desulphating enzyme in faeces could therefore be very important in determining the rate of degradation of secreted mucus and hence the level of protection of the mucosa. 2. A novel assay for mucin sulphatase has been developed using biologically labelled human colonic [35S]sulphomucin as a substrate and a mucin sulphatase has been purified from faeces by sequential high-performance gel filtration and ion-exchange chromatography. 3. The mucin sulphatase has been shown to have a pH optimum of 4.5 and activity over the pH range 3-7. It has a pI of 4.0 and is inhibited by inorganic sulphate and phosphate. The purified enzyme preparation gave a single band on electrophoresis with a molecular mass of 15,000 Da. It has a Km of 41.9 mmol/l and a Vmax. of 1.17 katal/kg for glucose 6-sulphate. The enzyme was also shown to enhance fivefold the deglycosylation of [3H]glucosamine-labelled mucin by a faecal mucin glycosidase preparation. 4. Two bacteroides spp. isolated from normal human faeces, Bacteroides fragilis and B. thetaiotaomicron, were found to be producers of mucin-desulphating enzymes. 5. Mucin sulphatase is likely to be critical in determining the rate of enzymic degradation of secreted colonic mucin.     

Postoperative analgesia and early rehabilitation after total knee replacement: A comparison of continuous low‐dose intravenous ketamine versus nefopam

The effects of nefopam and ketamine on pain control and rehabilitation after total knee replacement were compared in a prospective, double blinded study.Seventy-five patients were randomly assigned to receive a 0.2 mg kg^?1 bolus of nefopam or ketamine, followed by a 120 μg kg^?1 h^?1 continuous infusion until the end of surgery, and 60 μg kg^?1 h^?1 until the second postoperative day, or an equal volume of saline considered as placebo. Pain scores measured on a visual analog scale at rest and on mobilization, and patient-controlled intravenous morphine consumption, were assessed during 48 h. We measured the maximal knee flexion on the third postoperative day, and the delay to obtain a 90° flexion.Ketamine and nefopam reduced morphine consumption (p < 0.0001). Pain scores, were lower at rest and on mobilization in the ketamine group compared to the two other groups at all times of measurement. Pain score were lower in patients receiving nefopam compared to placebo, on arrival in the recovery room and at 2 h. Ketamine improved knee flexion on post operative day 3 (59° [33–63] vs. 50° [47–55] and 50° [44–55] in ketamine, placebo and nefopam groups, respectively, p < 0.0002) and decreased the delay to flex the knee at 90° (9.1 ± 4.2 vs. 12.3 ± 4.0 days, in ketamine and placebo groups, respectively, p = 0.01).Ketamine produces opioid-sparing, decreases pain intensity, and improves mobilization after total knee replacement. Nefopam achieves less significant results in that circumstances.     

Automated quantification of bone and liver alkaline phosphatase isoenzymes of human serum

Coupling two Technicon AAII samplers synchronised at 50 per hour with a 2 : 1 sample to wash ratio, sera are denatured and collected automatically. The incubation is done in continuous flow by passage through a U device made of large metallic needles soaked in a water bath at 60 ± 0.1°C. This allows a very quick temperature equilibration and a very reproducible incubation time of 35 sec. Initial and residual activities of alkaline phosphatase (ALP: EC 3.1.3.1) are measured on a Rotochem II (Aminco) with the procedure recommended by the Société Franlaise de Biologie Clinique (SFBC). For a mixture of bone and liver ALP, the initial rate constant of heat denaturation K_app = (A × K_b) + (B × K₁), where A and B are the fractions of each isoenzyme in the mixture, and K_b and K₁ the rate constants for bone (b) and liver (1) experimentally determined as 1.8 min^?1 and 0.45 min^?1 respectively. An equation was derived which converts the percent residual activity to a percentage of bone and liver isoenzyme: % bone ALP = 183 ? 2.38 ×% residual activity. This automated method was applied to 2700 people of both sexes from 4 to 100 years old.