Rapid measurement of total plasma homocysteine by HPLC

BACKGROUND: Determination of plasma homocysteine has gained increasing interest during the past few years. Several HPLC methods for determination of homocysteine are available. Based on these methods, we developed a new HPLC assay for rapid and sensitive measurement of total plasma homocysteine. METHODS: As a reducing reagent tris-(2-carboxylethyl)-phosphine is used, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate serves as the derivatization agent. Separation is performed by reversed-phase HPLC using a precolumn and a 55-mm RP(18) cartridge; mobile phase: 0.1 mol/l KH(2)PO(4) with 5% methanol, adjusted to pH 2.7 with ortho-phosphoric acid, flow-rate 1.1 ml/min. RESULTS: Homocysteine is clearly separated from other thiols, the retention time being 2.2 min, total analysis time is 6 min. Tests for linearity, recovery and precision are satisfactory, as well as the comparison with a commercial available assay method. Detection limit of the method is 0.5 micro mol/l, it could be further enhanced for measurements of even lower homocysteine concentrations in, e.g., cell culture supernatants. CONCLUSIONS: The described method is well suited for analysis of thiols in blood specimens. It is more convenient and more rapid than methods described earlier.     

Comparison of Abbott IMx total homocysteine assay with a high pressure liquid chromatography method for the measurement of total homocysteine in plasma and serum from a Norwegian population.

In this study 189 samples were analysed for the measurement of homocysteine by the Abbott IMx homocysteine assay and an HPLC method. A strong correlation was obtained between the homocysteine measurements performed by the Abbott IMx homocysteine assay and the HPLC method (coefficient of correlation, r2 = 0.947, p < 0.0001). The plot of the difference for the homocysteine measurements between the two methods against the average of the two measurements resulted a mean difference of 0.80 +/- 6.66 (mean +/- 2SD), and 0.008 +/- 0.126 (mean +/- 2SD) for log converted values of homocysteine. The concentrations of homocysteine measured in all the samples by the two methods were not significantly different. However, the Abbott IMx homocysteine assay measured the concentrations of total homocysteine in hyperhomocysteinemia as significantly higher than the HPLC method (25.00 micromol/l vs. 23.12 micromol/l, p < 0.0001). More studies may be required to explore factors that may influence measurements of homocysteine by the Abbott IMx homocysteine assay and the HPLC method.     

Correlates of plasma total homocysteine in patients with hyperlipidaemia

The study sought to define the relation of plasma total homocysteine to biological and clinical variables and to serum vitamin concentrations in patients with primary hyperlipidaemia. Fasting plasma total homocysteine was measured in 219 men and 159 women; vitamin concentrations were available for about 60% of the sample. Men had significantly higher plasma total homocysteine than women [median (25th, 75th percentiles) 9.4 (8.2, 11.5) μmol L⁻¹ vs. 8.5 (7.0, 10.2) μmol L⁻¹; P  = 0.0001]. Plasma total homocysteine was lower in women taking lipid-lowering drugs than in women who were not taking drugs. Serum folate and vitamin B₁₂ concentrations were normal for all but one and four subjects respectively. Correlations ( P  ≤ 0.06) were found between plasma total homocysteine and age, triglyceride concentration in women, uric acid concentration in men, serum folate, vitamin B₁₂ and creatinine concentrations. In multiple regression analysis, the association between plasma total homocysteine and sex and between plasma total homocysteine and use of lipid-lowering drugs disappeared when creatinine concentration was entered into the analysis. This study shows that plasma total homocysteine is related to vitamin concentrations within the normal range, suggesting that plasma total homocysteine may be modifiable by diet in hyperlipidaemic subjects with normal vitamin nutritition. Sex-related differences appear to be related to men's higher creatinine concentration. Whether lipid-lowering drugs interact with total homocysteine concentration requires further study.     

Influence of sodium fluoride on the stability of human blood samples and blood gas measurements

OBJECTIVE: This study aimed to reduce the analytical error associated with measuring oxygen and carbon dioxide partial pressures as well as the pH in arterial blood samples an hour after sample collection. The standard blood sample preparation procedure involving sample cooling down to 0 degrees C is known to have several drawbacks. Therefore, another approach using NaF at room temperature as an inhibitor of metabolic reactions was introduced. DESIGN AND METHODS: Arterial heparin blood samples from six volunteers were distributed over 104 single capillaries prepared with different concentrations of NaF. The capillaries were filled simultaneously and under the same conditions with blood samples, and the blood gas parameters of each sample were measured. Changes in pO2, pCO2, and pH during storage were evaluated with the aid of t test statistics. RESULTS: During the storage period under investigation, fluctuations of the carbon dioxide partial pressure and the pH were low, whereas there was a significant (P < 0.01) decrease of the oxygen partial pressure. This was observed at all NaF concentrations. Depending on the addition of NaF, a significant baseline shift for the time-resolved pH and pCO2 values could be observed. Whereas the partial pressure of carbon dioxide and the pH could be kept stable by adding a defined amount of NaF, the partial pressure of oxygen decreased significantly over 70 min. CONCLUSIONS: The proposed new method can be practically applied to a comparative blood gas study, significantly reducing the blood sample volume required. The application of analytical grade NaF is an improvement compared to previous work because a pH decrease could not be observed.     

Effects of sodium fluoride on total serum protein levels and transaminase activity in rats.

Transaminase activity and serum total protein level were investigated in adult rats after oral treating with sodium fluoride at three doses, 10, 20 and 30 mg/kg daily for 90 days. After 90 days, the average total serum protein level of the rats in the treatment group decreased significantly compared with that in the control [1.9 +/- 0.1 (mean +/- S.D., n = 140) vs. 3.1 +/- 0.2] mg/dl, P< 0.05. Serum transaminase activity in the treatment group increased compared with that in the control [5.3 +/- 0.4 (mean +/- S.D., n = 140) vs. 3.2 +/- 0.3] micromol/min per ml, P < 0.05.     

Stability of total plasma homocysteine in perinatology

BACKGROUND: Typical assay methods for total homocysteine in human plasma involve EDTA-containing whole blood. Unfortunately, rapid increases of the plasma homocysteine concentrations due to cellular export are observed when the EDTA-containing samples are not stored on ice and processed shortly after collection. This is a cumbersome procedure in perinatal settings, whereby delivery usually takes place at unpredictable times. METHODS: The stability of homocysteine was assessed from six placental and neonatal blood samples collected in citrate buffer. Samples were stored at 4 degrees C and tested at regular intervals for the first 24 h. RESULTS: There was no statistical difference in homocysteine concentrations as observed over the study storage period (mean coefficient of variation [CV] 4.9%). CONCLUSIONS: Citrated samples can be left in a refrigerator for at least 24 h with no effect on the plasma homocysteine concentrations.     

Clinical validation of a new blood collection tube for the accuracy of total homocysteine measurement by different methods

OBJECTIVE: To evaluate the clinical use of Homocysteine-Primavette, a new blood collection medium for total homocysteine (tHcy) assay. METHODS: The agreement between baseline tHcy and tHcy in stabilized samples (40 h) was assessed for FPIA, HPLC, GC-MS, LC-MS, and ICL. RESULTS: tHcy concentrations in whole blood were stable for 40 h in Hcy-Primavette tubes. CONCLUSION: Primavette tubes are a good alternative for the accurate tHcy measurement and no readjustment of reference intervals is needed.     

Postmenopausal osteoporosis: treatment with low-dose sodium fluoride and estrogen

Eleven menopausal patients were treated for 12 to 18 months with low-dose sodium fluoride and calcium. Six patients also received estrogen replacement. A significant increase in spine or hip bone mineral density measured by dual photon absorptiometry was observed in all patients. The estrogen-treated group had the greatest increase in bone density. Addition of estrogen seems to supplement bone gain and allow sodium fluoride to be administered in lower doses, which are easily tolerated and yet effective.     

Differential activation of rabbit femoral arteries by aluminum fluoride and sodium fluoride

The effect of fluoride (NaF; 10 mM sodium fluoride plus deferoxamine to chelate contaminating aluminum) and fluoride plus aluminum fluorides (AlF; 10 mM sodium fluoride plus 20 microM aluminum chloride) on activation of rabbit femoral arteries was investigated. AlF and NaF produced large increases in stress (force/muscle cross-sectional area), but temporal changes were dissimilar, as were other indices of muscle activation. Stress produced by NaF developed slowly and only after a long delay of about 15 min, whereas stress produced by AlF developed rapidly after a delay of only about 5 min. NaF-induced contractions were more sustained than AlF-induced contractions. Both AlF and NaF increased the level of cross-bridge phosphorylation and the velocity of muscle shortening, but at comparable stresses, AlF produced greater increases than did NaF. AlF produced a large increase in lP production, whereas NaF produced a small increase. Also, AlF-induced stress was largely insensitive to inhibition by the calcium channel blocker, nifedipine (1 microM), whereas NaF-induced stress was largely inhibited by nifedipine. However, in tissues depleted of calcium, both agents produced potent contractions when CaCl2 was added back to the tissues (EC50 values for AlF, NaF, histamine, phenylephrine and KCl were, respectively, 0.057, 0.085, 0.11, 0.11 and 0.23 mM). AlF, but not NaF, strongly desensitized arteries to phenylephrine, causing a 73% reduction in the ability of phenylephrine to achieve maximum steady-state stress. These data suggest that fluoride contracted rabbit femoral arteries by stimulating L-type calcium channels, and that aluminum fluoride stimulated phospholipase C, producing additional muscle activation.     

The measurement of complexed prostate-specific antigen has a better performance than total prostate-specific antigen.

The aim of this study was to compare the diagnostic utility of complexed prostate-specific antigen (cPSA) with total PSA (tPSA) in screening for prostate cancer. Serum concentrations of tPSA and cPSA were measured in 4479 adult men during the prostate cancer screening program in the Saarland region (Germany). The percentage of men with c/tPSA ratio above the cut-off value of 0.75 increased with increasing tPSA intervals: tPSA 0-0.9 microg/l, 4.4%; 1.0-1.9 microg/l, 24.3%; 2.0-2.9 microg/l, 43.9%; 3.0-3.9 microg/l, 50.4%; and 4.0-20 microg/l, 60.2%. The commonly accepted tPSA cut-off value of 3.9 microg/l matched to the 93rd percentile of the overall population (corresponding cPSA value, 2.9 microg/l). A total of 202 men out of 313 with increased cPSA had increased c/tPSA ratio (cut-off > or = 0.75) vs. 186 out of 312 men with increased tPSA. Thus, an additional 16 men at high risk for prostate cancer were selected only if cPSA was utilised as a first line parameter. Our data show that, compared to tPSA, cPSA measurement will always detect more high-risk patients, independent of the cut-off levels utilised for cPSA, tPSA and c/tPSA ratio. cPSA is more effective than tPSA in selecting subjects with an elevated c/tPSA ratio who are at high risk of prostate cancer. Thus, cPSA might be seen as the superior first-line parameter in screening for prostate cancer. Using lower cut-off values for tPSA or cPSA than the commonly accepted values seems reasonable for screening purposes.     

Assessing plasma total homocysteine in patients with end-stage renal disease.

Elevated plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease; however, in light of several recent randomized trials, the issue of causality has been cast into doubt. Patients with end-stage renal disease are particularly interesting as they consistently have elevated tHcy and their leading causes of morbidity and mortality are related to cardiovascular disease. In the present article, we review the early evidence for the homocysteine theory of atherosclerosis, homocysteine metabolism, mechanisms of toxicity, and pertinent available clinical investigations. Where appropriate, the sparse evidence of homocysteine in peritoneal dialysis is reviewed. We conclude by addressing the difficulties associated with lowering plasma tHcy in patients with end-stage renal disease and suggest some novel methods for lowering tHcy in this resistant population. Finally, to address the issue of causality, we recommend that clinicians and scientists await the results of the FAVORIT trial before abandoning homocysteine as a modifiable risk factor for cardiovascular disease, as this study has recruited patients from a population with consistently elevated plasma tHcy who are known to respond to vitamin therapy.     

同型半胱氨酸在糖尿病视网膜病变诊断中的应用

目的评价同型半胱氨酸（homocysteine,Hcy）测定在糖尿病视网膜病变（DR）诊断中的应用。方法应用循环酶法检测DR 45例、糖尿病无视网膜病变（NDR）50例及健康体检者40例血清Hcy含量,对结果进行统计分析。结果各组血清Hcy含量及高同型半胱氨酸（HHcy）发生率分别为：正常对照组（8.96±2.92）μmol/L、5.0%;NDR组（9.09±3.62）μmol/L、6.0%;DR组（21.82±7.99）μmol/L、73.3%;DR组HHcy血症发生情况及血清Hcy水平显著高于正常对照组及NDR组（P〈0.01）,而NDR组与正常对照组比较差异无统计学意义（P〉0.05）。结论血清Hcy水平升高也许是DR的危险因素之一,检测糖尿病患者血清Hcy水平有助于DR的早期诊断和治疗。     

草酸钠-氟化钠采血管中血样离心前放置时间对血浆葡萄糖测定的影响

目的探讨草酸钠-氟化钠采血管中血样在离心前放置的时间对血浆葡萄糖测定结果产生的影响。方法收集102例志愿者的血样,将同一患者标本分别置于4管草酸钠-氟化钠采血管和4管带促凝剂分离胶采血管内,草酸钠-氟化钠采血管血样在放置0、30、60、120min后离心,带促凝剂分离胶采血管血样凝固后离心,分别放置0、30、60、120min后和草酸钠-氟化钠采血管一同测定葡萄糖浓度。结果草酸钠-氟化钠采血管中血样的血浆葡萄糖浓度随离心前放置时间增加而降低,带促凝剂分离胶采血管中样本葡萄糖浓度变化不具有统计学意义。结论草酸钠-氟化钠采血管中血样离心前放置时间对血浆葡萄糖测定结果有影响,放置时间增加会导致测定值降低。