RESUMEN
A simple and senmsitive micromethod based on HPLC is described for the measurement of diclofenac in 200 ul plasma. A single extraction with dichlormethane in acidic medium was an essential clean-up step. Diclofenac and its internal standard (cyclohexendiphenyl propionic acid) were eluted at 3.3 and 6.5 min from a 4-micron C18 reverse-phase column using a mobile phase consisting of 0.75 M sodium acetate buffer, pH 5.0, and acetonitrile (55:45, v/v) at a flow rate of 0.9 ml/min with detection at 282 nm. The method, validated on the basis of parameters evaluated nfor the confidence limits of diclofenac measurements in spiked plasma, presented 1 ng/ml sensitivity, 10-10,000 ng/ml linearity, and 3.5% and 5.7% intra-and interassay precision, respectively. Peak plasma diclofenac levels ranging from 177 to 841 ng/ml and from 276 to 1008 ng/ml were obtained for two slow-release formulations. A wide range (1 ng/ml-3 ug/ml) was observed for plasma diclofenac levels of volunteers during a 24-h study period
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Diclofenaco , Cloruro de Metileno , Plasma/análisisRESUMEN
Se evaluó el estado nutricional de zinc y cobre en 40 embarazadas con índice de peso normal del Area Norte de Santiago. Las madres del Grupo Control (21) recibieron atención regular de Consultorio mientras que las del Experimental (19) recibieron además, 20 mg de sulfato de zinc, diariamente, desde la semana 25. A las 13 y 37 semanas de gestación, se realizó una encuesta alimentaria y se determinó zinc y cobre en suero. La ingesta global fue insuficiente en ambos controles, especialmente la de los nutrientes estudiados (más del 70% de las madres tenían un consumo <75% de la recomendación). Los valores plasmáticos promedio fueron normales, con disminución del zinc y aumento del cobre en el control final. Sin embargo, un 22,5% tuvo valores de zinc deficientes, aumentando a 42,9% al término en el grupo no suplementado (p< 0,05). La deficiencia de cobre fue muy baja en ambos controles sin diferencias entre los grupos. No se registraron anomalías del embarazo, parto ni recién nacidos. El escaso efecto clínico de la deficiencia de estos nutrientes así como el mínimo efecto bioquímico de una suplementación con zinc sugiere la necesidad de utilizar indicadores más sensibles para evidenciar el déficit.
Asunto(s)
Embarazo , Humanos , Femenino , Cobre/deficiencia , Tercer Trimestre del Embarazo , Zinc/deficiencia , Plasma/análisis , Zinc/fisiologíaRESUMEN
Foi proposto um metodo para determinacao de fenobarbital em plasma de pacientes submetidos a terapia antiepiletica. O metodo inclui uma extracao com cloroformio seguida de purificacao com n-hexano e acetonitrila. A quantificacao do fenobarbital foi feita por cromatografia em fase gasosa (CGL), detector de ionizacao de chama (DIC) e coluna empacotada GP 2% SP-2110/1% SP 2510-DA. O coeficiente de variacao (3,8 a 9%) e a sensibilidade do metodo (1 micro g/ml plasma) mostraram-se adequados para detectar o farmaco em concentracoes subterapeuticas, sem a necessidade de derivacao
Asunto(s)
Anticonvulsivantes/uso terapéutico , Fenobarbital/análisis , Plasma/análisis , Cromatografía de Gases , Cromatografía Liquida/métodosRESUMEN
Detection and quantification of bacterial endotoxin in plasma by the Limulus amebocyte lysate test (or other assays for endotoxins) is hindered by the presence of inhibitors. Treatment of plasma to overcome inhibitory activities is required before plasma can be successfully assayed for endotoxin. We have conducted an investigation comparing the three most commonly used procedures (dilution-heating, trifluoroacetic acid oxidation, and chloroform extraction) for treatment of plasma before its assay for endotoxin with the chromogenic Limulus test. Initially, conditions were optimized for treatment of plasma by each of these methods. Subsequently, a direct comparison of the three plasma treatment procedures was performed with plasma spiked with known concentrations of endotoxin. The optimized dilution-heating procedure resulted in the most sensitive detection of endotoxin, with sensitivity approximately 10 times greater than the optimized trifluoroacetic acid oxidation procedure and approximately 100 times greater than treatment of plasma by chloroform extraction. Maximal detection of low concentrations of endotoxin by the chromogenic Limulus test was obtained by dilution of plasma fourfold with 0.15 mol/L NaCl followed by heating at 60 degrees C for 30 minutes. This procedure was simple, rapid, and did not involve addition of any reagents to plasma that could potentially add contaminating endotoxin.
Asunto(s)
Endotoxinas/sangre , Plasma/análisis , Salmonella , Animales , Técnicas Bacteriológicas , Cloroformo , Humanos , Prueba de Limulus , Plasma/metabolismo , Plasma/microbiología , Salmonella/aislamiento & purificación , Salmonella/metabolismo , Cloruro de Sodio , Ácido TrifluoroacéticoRESUMEN
This paper describes the analysis, by a highly sensitive and specific enzyme immunoassay (EIA), of AcSDKP, a tetrapeptide recently isolated from fetal calf bone marrow and subsequently purified and identified which substantially inhibits entry into cycle of hematopoietic pluripotent stem cells (CFU-S). This molecule has a marked protective effect in mice during anticancer chemotherapy with phase-specific drugs and plays an essential role in maintaining CFU-S out of cycle in normal mice. Using acetylcholinesterase-AcSDKP conjugate as tracer, rabbit specific antiserum and 96-well microtiter plates coated with a mouse monoclonal anti-rabbit IgG antibody, this EIA allows detection of AcSDKP at 15 fmol levels with a coefficient of variation less than 10% in the 50-500 fmol range. When combined with high-performance liquid chromatography, this assay clearly reveals the presence of this peptide in normal human white blood cells whereas in supernatant from cultured lymphocytes and in plasma the immunoreactive material is distinct from standard AcSDKP.
Asunto(s)
Células Madre Hematopoyéticas/citología , Técnicas para Inmunoenzimas , Leucocitos/análisis , Oligopéptidos/sangre , Plasma/análisis , Acetilcolinesterasa , Secuencia de Aminoácidos , Especificidad de Anticuerpos , División Celular/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Oligopéptidos/farmacologíaRESUMEN
Fresh-frozen plasma (FFP) and cryoprecipitate both contain Factors I and VIII, however thawed FFP may be stored at 1-6 degrees C for 24 hours, but thawed cryoprecipitate may only be stored at 1-6 degrees C for six hours when used for Factor VIII content. To determine whether it would be safe and effective to extend the storage period of thawed cryoprecipitate from 6 to 24 hours, Factor VIII (and fibrinogen) levels were measured in 25 units of cryoprecipitates immediately on thawing and at 6 hours and 24 hours thereafter. The Factor VIII activity level decreased to 86% of the original activity level within 6 hours, but the drop between 6 and 24 hours was relatively small. Eighty percent of the original activity was still present at 24 hours after thawing. The fibrinogen level decreased to 87% of the original level within 6 hours but remained stable between 6 and 24 hours. Additional testing showed that fibrinogen levels remained stable between 6 and 74 hours. These data suggested that the storage of thawed cryoprecipitate might be extended to 24 hours when this blood product is used for Factor VIII content and to 74 hours when it is used for fibrinogen content. Furthermore, the lack of prohibition on the use of cryoprecipitate that has been thawed for more than six hours and stored at 4 degrees C for its fibrinogen content seems reasonable.
Asunto(s)
Conservación de la Sangre , Criopreservación , Factor VIII/análisis , Fibrinógeno/análisis , Plasma/análisis , Humanos , Factores de TiempoRESUMEN
Se evaluó el efecto del ejercio físico agudo sobre la distribución y el metabolismo de la vitamina A en un modelo experimental animal. Se estudiaron las concentraciones plasmáticas, hepaticas, renales, testiculares y de glándulas suprarrenales de la vitamina A y de sus principales formas moleculares: retinol y ésteres de retinol, en animales sometidos a ejercicio físico durante 2 h sin previo entrenamiento. Se observó una disminución en la concentración hepática y testicular de la vitamina A y un incremento de ésta en los riñones de los animales ejercitados. Se demostró, además, el incremento del catabolismo hepático del retinol bajo las condiones experimentales
Asunto(s)
Ratas , Animales , Masculino , Ejercicio Físico , Glándulas Suprarrenales/análisis , Hígado/análisis , Plasma/análisis , Ratas Endogámicas/metabolismo , Riñón/análisis , Testículo/análisis , Vitamina A/metabolismo , Cromatografía Líquida de Alta PresiónRESUMEN
Se determinaron las concentraciones de sodio y potasio en plasma sanguíneo, saliva y eritrocitos en 3 grupos de pacientes. Grupo A (n=27) normotensos sin antecedentes familiares de hipertensión arterial y grupo C (n=35), individuos que padecían de hipertensión arterial. Se encontró un aumento estadísticamente significativo (p < 0,05) del sodio y una disminución del potasio intraeritrocitario, así como un aumento del sodio y potasio en la saliva en el grupo C con respecto a los demás grupos estudiados. No hubo diferencias significativas entre los grupos A y B en ninguno de los parámetros.
Asunto(s)
Adolescente , Adulto , Persona de Mediana Edad , Humanos , Masculino , Femenino , Eritrocitos/análisis , Hipertensión/fisiopatología , Plasma/análisis , Potasio/análisis , Saliva/análisis , Sodio/análisisRESUMEN
It is the activity that determines the direction of chemical processes, transport, etc. and thus provides the clinically more relevant information. Direct reading glucose electrodes consume glucose at a rate proportional to the glucose activity in the sample. The activity equals the molality (mmol glucose per kg water), so results from direct reading glucose electrodes must differ from the conventionally measured glucose concentration. This was observed in 159 whole blood samples which gave higher results from a direct reading glucose electrode than by our conventional method (y = 1.21x - 0.37 mmol/l). However, adjustment for the different water concentration due to salt, plasma proteins, and hemoglobin occupying space, gave results equal to the concentrations (y = 1.00x - 0.28 mmol/l, r = 0.997). Furthermore, results for samples with constant glucose concentration and varying albumin concentration correlated with the albumin concentration (r = 0.989), but not after adjustment for water concentration (r = 0.037, n.s.).
Asunto(s)
Análisis Químico de la Sangre/instrumentación , Glucemia/análisis , Plasma/análisis , Electrodos , Eritrocitos/fisiología , Humanos , Concentración Osmolar , Análisis de Regresión , Albúmina Sérica/farmacologíaRESUMEN
Patients received 2,000 ml of dialysate intraperitoneally with five exchanges per day during continuous peritoneal dialysis (CAPD) for the treatment of terminal renal insufficiency. During a dwell time of 4 h the dialysate reached a total protein concentration up to 100 mg/dl by mass transfer of intravascular proteins. The composition is dependent on the molecular weight of the proteins. This results in an intraperitoneal hemostatic system of low concentration and different composition. We found an intraperitoneal fibrinogen cleavage and thrombin-antithrombin III-complex formation leading to increased levels of fibrinopeptide A (FPA: 33.3 +/- 7.0 ng/ml) and thrombin-antithrombin III-complex (TAT: 4.7 +/- 0.4 ng/ml) in plasma by mass transfer from dialysate to plasma. t-PA (tissue plasminogen activator) and PAI-1 (plasminogen activator inhibitor type 1) concentrations in plasma were within the normal range. The dialysate concentrations indicated a low local secretion. The fibrinolytic fibrin fragment D-dimer and the fibrinogen degradation product concentrations in plasma were greater than in dialysate. But the relations of the proteins between plasma and dialysate refer to a local intraperitoneal production as well. The results show that intraperitoneal coagulation predominates over fibrinolysis which is accompanied by an intravascular fibrinolysis in patients undergoing CAPD. Neoantigens produced in dialysate and diffused to plasma are comparable to changes seen in disseminated intravascular coagulation.
Asunto(s)
Líquido Ascítico/análisis , Coagulación Sanguínea/fisiología , Fibrinólisis/fisiología , Diálisis Peritoneal Ambulatoria Continua/efectos adversos , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Plasma/análisisRESUMEN
In order to determine the effects of large variations in plasma amino acid concentrations upon human erythrocyte amino acid content, the plasma concentration of blood samples was enhanced (x 3.8) by adding amino acids or decreased (x 0.49) by plasma dilution. Before and after incubation (30 s at 37 degrees C), the erythrocyte contents were calculated from whole blood and plasma amino acid concentrations. Large and rapid plasma concentration variations led to significant erythrocyte changes in 11 amino acids. THR, CIT, alpha AB, VAL, MET, ILE, LEU, TYR, PHE, TRP, and ARG. Relationships between erythrocyte and plasma concentrations were determined for these amino acids. These observations were examined in the light of the role played by erythrocytes in blood amino acid transport.
Asunto(s)
Aminoácidos/sangre , Eritrocitos/análisis , Plasma/análisis , Humanos , Análisis de RegresiónRESUMEN
The effect of varying the type of column and eluent composition on drug-free plasma profiles was investigated. The study was based on a C18 and a CN column; methanol and acetonitrile were the organic modifiers used. The plasma profiles were evaluated quantitatively by measuring the number of interfering peaks greater than 8 . 10(-4) absorbance units in the area of interest along the chromatogram. Results were subjected to statistical treatment using a three-factor analysis of variance design. The three factors were the column, the type of organic modifier and either the percentage organic modifier, the pH or the ionic strength. Analysis of the data revealed that significant effects were seen with changing eluent composition, particularly with regard to the percentage of organic modifier, and that the observed effects were strongly dependent on the type of column and the type of organic modifier under consideration.
Asunto(s)
Plasma/análisis , Solventes , Acetonitrilos , Análisis de Varianza , Cromatografía Líquida de Alta Presión/métodos , Concentración de Iones de Hidrógeno , Metanol , Espectrofotometría UltravioletaRESUMEN
A high yield, intermediate purity factor VIII concentrate derived from heparinized plasma has been developed which can be heat-treated at 60 degrees C, 68 degrees C or 80 degrees C/72 h to permit inactivation of viral contaminants which may be present. After cold reprecipitation of the heparinized cryoprecipitate (CRC), the resolubilized CRC precipitate was adjusted to 25-30 mg/ml protein and pH 6.35 +/- 0.1 and incubated for 1 h at 8 degrees C. After centrifugation to remove the precipitated fibrinogen and fibronectin, a factor VIII-rich supernatant can be recovered which contains greater than 500 units of VIII:C per liter of starting plasma (Method I product) at a purity of 1.5 U/mg protein. Adjusted to 50 mM glycine and pH 6.8, the product can be lyophilized and heat-treated at 60 degrees C/72 h without a significant loss of VIII:C activity. However, at 68 degrees C or 80 degrees C/72 h, temperatures now reported to be more effective in viral inactivation, the recoveries were reduced to 68 and 33% respectively. Significantly improved recoveries after heat-treatment (HT) at 68 degrees C or 80 degrees C/72 h were achieved if the 8 degrees C supernatant product was prepared by a modified procedure (Method II). This further reduces the fibrinogen content of the product while maintaining VIII:C yields greater than 500 U/l at a purity of 1.9 U/mg. When adjusted to 50 mM glycine and 1-2% (w/v) sucrose (pH 6.8), lyophilized and heat treated at 60 degrees C, 68 degrees C or 80 degrees C/72 h, the VIII:C recoveries of Method II product were 88-100%, 79-84% and 80-83% of pre-HT levels respectively. The yield of VIII:C was greater than 400 U/l at a purity of 1.6-1.4 U/mg at 1-2% (w/v) sucrose even after the severe heat-treatment at 80 degrees C. In addition, the von Willebrand factor multimers are similar in size and triplet pattern to those observed in routine cryoprecipitate preparations.
Asunto(s)
Factor VIII/aislamiento & purificación , Factor de von Willebrand/aislamiento & purificación , Precipitación Química , Frío , Liofilización , Heparina , Calor , Humanos , Sustancias Macromoleculares , Plasma/análisis , Plasma/efectos de los fármacos , Solubilidad , Agua/análisisRESUMEN
A novel enzyme-linked immunosorbent assay for the quantitation of elastase linked to alpha 2-macroglobulin (alpha 2M) (elastase-alpha 2M complex, EMC) in body fluids is presented. The assay has a lower detection limit of 1.5 ng bound elastase per ml. The critical factor allowing immunological detection of alpha 2M-bound elastase is the addition of phenyl methyl sulphonyl fluoride (PMSF) to the assay buffer. The assay has been used to quantitate EMC in plasma and serum of healthy volunteers, and assess the distribution of elastase between its two main inhibitors, alpha 2M and alpha 1-proteinase inhibitor (API). EMC levels in serum samples from volunteers were significantly higher than in plasma (26.9 vs 21.9 ng/ml, p = 0.05, Student's t-test for paired samples). The API-bound elastase levels were 70.2 and 170.1 ng/ml in plasma and serum respectively (statistically significantly higher in serum, p less than 0.0001). The ratio of macroglobulin to alpha 1-proteinase-bound elastase levels was 0.333 in plasma and 0.168 in serum (p less than 0.0001). As alpha 2M bound elastase retains some proteolytic activity, direct measurement of circulating levels in inflammatory conditions should be of interest.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Elastasa Pancreática/sangre , alfa 1-Antitripsina/metabolismo , alfa-Macroglobulinas/metabolismo , Cromatografía en Gel , Etanol , Humanos , Fluoruro de Fenilmetilsulfonilo/farmacología , Plasma/análisisRESUMEN
A specific high-performance liquid chromatographic (HPLC) method using precolumn derivatization and fluorescence detection was developed for the determination of the monoamine oxidase B inhibitor Ro 19-6327 in human plasma. After extraction of the basified plasma with tert.-butyl methyl ether-1-butanol (8:2, v/v) and back-extraction into dilute phosphoric acid, the solution was neutralized with phosphate buffer and the drug derivatized with fluorescamine. The derivative was chromatographed on a reversed-phase C8 column, using phosphate buffer-acetonitrile (68:32, v/v) as mobile phase, and fluorescence detection (excitation 370 nm, emission 485 nm). The limit of quantification was 1 ng/ml using 1 ml of plasma. The recovery was 79% in the range 5-200 ng/ml and the inter-assay precision was 3.1-7.9% in the range 2-500 ng/ml. The compound proved to be stable in human plasma. Moderate instability was found in rat plasma and, surprisingly, severe instability in dog plasma. Measures for handling unstable dog plasma samples are described. An HPLC method with UV detection was used for the analysis of dog and rat plasma samples, which is also described briefly. The fluorescence method, which was five times more sensitive than the UV method, was successfully applied to a human tolerance study.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Fluorescencia , Inhibidores de la Monoaminooxidasa/sangre , Ácidos Picolínicos/sangre , Plasma/análisis , Fluorescamina , HumanosRESUMEN
Improvements in a multidimensional liquid chromatography system for the direct determination of drug substances in blood plasma are reported. The system employs an on-line micellar chromatographic cleanup followed by a reversed-phase analytical separation. The limit of detection of propranolol is improved by a factor 10 compared to previously reported work. The technique is applied towards the determination of a multicomponent mixture of tricyclic anti-depressants in blood plasma. A protocol for optimization is described.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Preparaciones Farmacéuticas/análisis , Plasma/análisis , Cromatografía Líquida de Alta Presión/instrumentación , Humanos , Propranolol/sangreRESUMEN
In order to clarify the relative contributions of activated platelets and other sources to circulating osteonectin, the amounts of osteonectin present in serum and in plasma prepared without significant platelet activation were compared to platelet count in normal subjects and patients with various degrees of thrombocytopenia. Serum osteonectin showed a logarithmic positive correlation with platelet count (r = 0.87, P less than 0.0001, n = 52). Osteonectin concentration was significantly lower in plasma than in matched sera in all subjects sampled, and not significantly different in plasma of thrombocytopenic and normal subjects. These results confirm the major contribution of platelets to serum osteonectin. The positive zero intercept of the plot osteonectin vs. platelet count (19 +/- 2.45 ng/ml, mean +/- SD, P less than 0.0001, n = 52) and the presence of significant amounts of osteonectin in plasma, document the existence of a basic level of circulating osteonectin, independent of platelet activation, and to which the relative contribution of bone remains to be assessed.
Asunto(s)
Osteonectina/sangre , Trombocitopenia/sangre , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Plasma/análisis , Recuento de PlaquetasRESUMEN
Plasma and erythrocyte (RBC) tocopherol-isomer concentrations were determined serially in forty-two premature infants (25-35 weeks gestation) from birth to 8 weeks of age. For comparison purposes vitamin E status was also determined in six term infants over the first 8 d following birth and in a group of thirteen adult volunteers. Vitamin E intakes in term and preterm infants were calculated from recorded food intakes and blood transfusions. In term infants plasma vitamin E concentration rose from 1.9 mg/l (day 1) to 8.2 mg/l by day 8. In comparison preterm plasma vitamin E concentration, 0.3 mg/l (day 1), did not change appreciably by day 8 (0.7 mg/l). Likewise RBC vitamin E concentration increased in term infants from 1.3 mg/l (day 1) to 2.7 mg/l (day 8), while in preterm infants it remained unchanged, 1.5 mg/1 (day 1) v. 1.3 mg/l (day 8). Over the 3 weeks following birth, RBC vitamin E concentrations in the premature infants increased to adult values, while plasma vitamin E concentration did not reach the adult range until 8 weeks post-term. These slow changes in plasma vitamin E status occurred even though the vitamin E intake of these infants was similar to that proving adequate for term infants.
Asunto(s)
Recien Nacido Prematuro/sangre , Vitamina E/sangre , Adulto , Eritrocitos/análisis , Eritrocitos/metabolismo , Humanos , Recién Nacido , Isomerismo , Plasma/análisis , Factores de Tiempo , Vitamina E/administración & dosificación , Vitamina E/metabolismoRESUMEN
Advances in the preparation of commercial Factor VIII concentrates have decreased the clinical use of cryoprecipitate to replace Factor VIII coagulant activity. Cryoprecipitate is now frequently transfused as a source of fibrinogen or von Willebrand factor (vWF). The minimum acceptable content of Factor VIII is prescribed, but no attempt is made to optimize, standardize, or assess the content of fibrinogen or vWF in cryo. If reasonably accurate information on the composition of cryoprecipitate were available, the physician could calculate an appropriate dose of cryo, thus avoiding unnecessary donor exposures and waste of product. This study was designed to measure the functionally active vWF and fibrinogen in cryoprecipitate prepared by three techniques in an attempt to optimize the yield of these hemostatically important components, and to obtain accurate information on the composition of the product. Cryoprecipitate was made from 82 units of fresh frozen plasma (FFP) as follows: (1) most of the supernatant plasma was expressed from "dry" cryo; (2) about 15 mL of plasma was left in each "regular" unit; and (3) "overnight" units were made from FFP thawed overnight in a refrigerator rather than in a 4 degrees C water bath as for the dry and regular units. Dry, regular, and overnight units had volumes of 8.8 +/- 1.5, 16.6 +/- 3.9, and 15.1 +/- 2.4 mL/bag, respectively. Dry cryoprecipitate units had significantly less fibrinogen and vWF than regular or overnight units. The vWF multimer pattern for all three types of cryoprecipitate was indistinguishable from that of normal pooled plasma. Thus, the amount of plasma expressed during preparation has a significant impact on the vWF and fibrinogen content of the resulting product. The amounts of these clinically important proteins should be assayed as a step toward rational determination of optimal cryoprecipitate doses in specific clinical settings.
Asunto(s)
Fibrinógeno/análisis , Plasma/análisis , Factor de von Willebrand/análisis , Conservación de la Sangre , Criopreservación , HumanosRESUMEN
The concentration of high-density lipoprotein cholesterol (HDL-C) in plasma is now established as an independent risk factor for coronary heart disease, but more data are needed on the relative risk-predictive powers of different HDL subclasses. For epidemiologic and clinical purposes, isolation of HDL from other lipoproteins and separation of its two major subclasses, HDL2 and HDL3, are performed most conveniently by precipitation. Although storage of plasma is commonly necessary, little information is available on the long-term stability of HDL subclasses at different temperatures. Therefore, we quantified HDL-C, HDL2-C, and HDL3-C by dual precipitation with heparin-MnCl2/15-kDa dextran sulfate (H-M/DS) in samples of EDTA-plasma from 93 healthy subjects, after storage for one to 433 days at -20 degrees C, at -70 degrees C, or in liquid nitrogen (-196 degrees C). Fourteen samples (15%) were stored for a year or longer. At -20 degrees C, HDL-C decreased by 4.8% per year and HDL3-C decreased by 6.9% per year (P = 0.002 for both variables) relative to results obtained with samples stored in liquid nitrogen; total cholesterol, HDL2-C, and triglyceride did not change significantly at this temperature. When stored at -70 degrees C, none of the lipids showed any change relative to results obtained with liquid nitrogen. Thus, long-term storage of EDTA-plasma at -20 degrees C is unsuitable for subsequent quantification of HDL-C and its subclasses by H-M/DS dual precipitation. Storage at -70 degrees C is preferable, and is as reliable as storage in liquid nitrogen.