An Assessment of the Serum Activity of ADH and ALDH in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC; previously known as primary biliary cirrhosis) is a chronic inflammation-induced cholestatic process in the liver. Antimitochondrial antibodies (AMAs) are observed in around 90% of patients, which suggests that PBC is an autoimmune disease. Alcohol dehydrogenase (ADH), ADH isoenzymes and aldehyde dehydrogenase (ALDH) are localized in the liver, and they are useful markers of liver dysfunction. In this study, the activity of total ADH, ADH isoenzymes and ALDH was evaluated in the blood serum of patients with PBC. The experimental group comprised 50 PBC patients, both male and female, aged 28-67. The control group consisted of 50 healthy subjects, both male and female, aged 25-65. The serum activity of class I ADH, class II ADH and ALDH was measured by spectrofluorophotometry, whereas total ADH and class III ADH activity was determined by photometry methods. The activity of class I ADH and total ADH was significantly higher in the experimental group than in the control group (p < 0.001). An increase in class I ADH and total ADH activity indicates that the isoenzyme class I ADH is released by compromised liver cells and can be useful diagnostic markers of PBC.

Activity of Total Alcohol Dehydrogenase, Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in the Serum of Patients with Alcoholic Fatty Liver Disease.

Background and objectives: The aim of the current study was to assess the use of determinations of total alcohol dehydrogenase and the activity of its isoenzymes as well as aldehyde dehydrogenase in the serum of patients with alcohol liver disease. Materials and Methods: The testing was performed on the serum of 38 patients with alcoholic fatty liver (26 males and 12 females aged 31-75). The total activity of ADH was determined by the colorimetric method. The activity of ADH I and ADH II, as well as ALDH, was determined by the spectrofluorometric method using fluorogenic specific substrates. The activity of isoenzymes of other classes was determined by spectrophotometric methods using substrates. Results: A statistically significantly higher ADH I activity was noted in the serum of patients with alcoholic fatty liver (4.45 mIU/L) compared to the control group (2.04 mIU/L). A statistically significant increase in the activity was also noted for the class II alcohol dehydrogenase isoenzyme (29.21 mIU/L, control group: 15.56 mIU/L) and the total ADH (1.41 IU/L, control group: 0.63 IU/L). Conclusions: The obtained results imply the diagnostic usefulness of the determination of AHD total, ADH I, and ADH II activity in the serum of patients with alcoholic fatty liver.

LC-MS/MS method for the differential diagnosis of treatable early onset inherited metabolic epilepsies.

Rapid diagnosis and early specific treatment of metabolic epilepsies due to inborn errors of metabolism (IEMs) is crucial to avoid irreversible sequalae. Nowadays, besides the profile analysis of amino- and organic acids, a range of additional targeted assays is used for the selective screening of those diseases. This strategy can lead to long turn-around times, repeated sampling and diagnostic delays. To replace those individual targeted assays, we developed a new liquid chromatography mass spectrometry method (LC-MS/MS) for the differential diagnosis of inherited metabolic epilepsies that are potentially treatable. The method was developed to simultaneously quantify 12 metabolites (sulfocysteine, guanidinoacetate, creatine, pipecolic acid, Δ1 -piperideine-6-carboxylate (P6C), proline, Δ1 -pyrroline-5-carboxylate (P5C), and the B6 -vitamers) enabling the diagnosis of nine different treatable IEMs presenting primarily with early-onset epilepsy. Plasma and urine samples were mixed with internal standards, precipitated and the supernatants were analyzed by LC-MS/MS. In comparison with previous assays, no derivatization of the metabolites is necessary for analysis. This LC-MS method was validated for quantitative results for all metabolites except P6C and P5C for which semiquantitative results were obtained due to the absence of commercially available standards. Coefficients of variation for all analytes were below 15% and recovery rates range between 80% and 120%. Analysis of patient samples with known IEMs demonstrated the diagnostic value of the method. The presented assay covers a selected panel of biochemical markers, improves the efficiency in the laboratory, and potentially leads to faster diagnoses and earlier treatment avoiding irreversible damage in patients affected with IEMs.

The Alterations in Alcohol Dehydrogenase Activity in the Sera of Women With Intrahepatic Cholestasis of Pregnancy.

BACKGROUND/AIM: The liver of pregnant women undergoes physiological and pathological changes and the changes in liver enzyme activity and release reflect changes in serum enzymatic activity. We aimed to assess the activity of alcohol dehydrogenase (ADH) isoenzymes, and aldehyde dehydrogenase (ALDH) in the sera of women with intrahepatic cholestasis of pregnancy (ICP), the most common pregnancy-related liver disease. PATIENTS AND METHODS: Serum samples were taken from 40 women with ICP in the second or third trimester of pregnancy. Serum samples were also obtained from 40 healthy pregnant women at the same time of pregnancy and 40 healthy non-pregnant women. Class I and II of ADH and ALDH activity was measured by a spectrofluorometric method. Class III, IV ADH and total ADH activity was measured by photometric methods. RESULTS: The total ADH activity was significantly higher in women with ICP than in healthy pregnant and non-pregnant women (about 42%). The median total activity of ADH was 1067 mU/l in women with ICP, 628 mU/l in healthy pregnant and 605 mU/l in non-pregnant women. A statistically significant increase in class I ADH isoenzymes was found in the sera of pregnant women with ICP. The median activity of this class in the ICP group increased about 62% and 80% in comparison to the healthy pregnant women and non-pregnant women, respectively. CONCLUSION: The activity of class I ADH isoenzymes in the sera of women with ICP is statistically significantly increased and may have a diagnostic significance.

Secondary metabolites of petri-dish cultured Antrodia camphorata and their hepatoprotective activities against alcohol-induced liver injury in mice.

Antrodia camphorata, a well-known and highly valued edible medicinal mushroom with intriguing activities like liver protection, has been traditionally used for the treatment of alcoholic liver disease. A. camphorata shows highly medicinal and commercial values with the demand far exceeds the available supply. Thus, the petri-dish cultured A. camphorata (PDCA) is expected to develope as a substitute. In this paper, nineteen triterpenes were isolated from PDCA, and thirteen of them were the unique anthroic acids in A. camphorata, including the main content antcin K, which suggested that PDCA produced a large array of the same anthroic acids as the wild one. Furthermore, no obvious acute toxicity was found suggesting the edible safety of PDCA. In mice alcohol-induced liver injury model, triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) had been reduced by the PDCA powder as well as the main content antcin K, which indicated that the PDCA could protect alcoholic liver injury in mice model and antcin K could be the effective component responsible for the hepatoprotective activities of PDCA against alcoholic liver diseases.

Evolutionary Changes in the Content of Dehydrogenase System Metabolites as a Mechanism of Adaptation to Cold in Higher Vertebrates.

It has been found that the optimal body balance control under the conditions of the adaptation to cold is mostly determined by the ratio of the blood concentrations of endogenous ethanol and acetaldehyde related to the activities of liver alcohol dehydrogenase and aldehyde dehydrogenase in the order of increasing level of adaptation: higher vertebrates unadapted to cold, including human → small animals adapted to cold → large animals adapted to cold native to the North → hibernators, regardless of the species specificity and the environment.

Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase Activity in the Serum of Patients with Non-alcoholic Fatty Liver Disease.

BACKGROUND/AIM: Non-alcoholic liver disease (NAFLD) is one of the most common causes of chronic liver disease, and its prevalence and medical importance is increasing worldwide. Changes in enzyme activity in liver cells in various liver diseases are reflected by an increase in serum enzymatic activity. For example, alcohol dehydrogenase activity (ADH) and aldehyde dehydrogenase (ALDH), that occur in the liver in large quantities, correlate with disease severity during cirrhosis. In the current study, the activity of ADH isoenzymes and ALDH in the serum of patients with NAFLD was investigated. MATERIALS AND METHODS: Serum samples were collected for routine biochemical studies from 55 patients with NAFLD patients and from 50 healthy individuals. Class I and II ADH and ALDH activity were measured by spectrofluorometric method. Photometric methods were used to measure ADH class III, IV and total ADH activity. RESULTS: Total ADH activity was significantly higher in non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) than in healthy individuals (44 and 48.5% activity, respectively). The median total activity of ADH was 1,164 mU/l in patients with NAFLD, 1,258 mU/l in NASH and 648 mU/l in the control group. The increase in ADH class I and II isoenzyme in serum of patients with NAFL and NASH was statistically significant. The activity of ADH I, ADH II, and total ADH significantly increased with increasing disease progression. CONCLUSION: The activity of isozymes of class I and II alcohol dehydrogenase in patients with NAFLD is enhanced and appears to be due to the release of these isoenzymes from damaged hepatocytes.

The Activity of Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in the Sera of Patients with Autoimmune Hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a progressive inflammatory hepatopathy and an important cause of end-stage liver. The liver cells' destruction is reflected by increased activity of different enzymes in the serum. These enzymes include alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which play a significant role in the metabolism of many biological substances and exist mainly in the liver. In this study we investigated the activity of alcohol dehydrogenase and its isoenzymes and the total activity of ALDH in the sera of patients with autoimmune hepatitis. METHODS: Serum samples were taken for routine biochemical investigation from 32 patients with autoimmune hepatitis and from 40 healthy subjects. Class I and II of ADH and ALDH activity was measured by the spectrofluorometric method. For measurement of class III ADH and total ADH activity we employed the photometric methods. RESULTS: The activity of the class I ADH isoenzyme was significantly higher in the sera of patients with autoimmune hepatitis. The median activity of this isoenzyme in the patients group was approximately 63% (3.94 mU/L) higher than the control level (1.46 mU/L). For this reason, the total ADH activity was also significantly increased. The activities of other ADH isoenzymes and ALDH tested were unchanged. CONCLUSIONS: The activity of total ADH and class I isoenzymes in the sera of patients with autoimmune hepatitis is increased, and it seems to be caused by the release of alcohol dehydrogenase from damaged liver cells.

The Alcohol Dehydrogenase Isoenzyme as a Potential Marker of Pancreatitis.

BACKGROUND/AIM: Human pancreas parenchyma contains various alcohol dehydrogenase (ADH) isoenzymes and also possesses aldehyde dehydrogenase (ALDH) activity. The altered activities of ADH and ALDH in damaged pancreatic tissue in the course of pancreatitis are reflected in the human serum. The aim of this study was to investigate a potential role of ADH and ALDH as markers for acute (AP) and chronic pancreatitis (CP). PATIENTS AND METHODS: Serum samples were collected for routine biochemical investigations from 75 patients suffering from acute pancreatitis and 70 patients with chronic pancreatitis. Fluorometric methods were used to measure the activity of class I and II ADH and ALDH activity. The total ADH activity and activity of class III and IV isoenzymes were measured by a photometric method. RESULTS: There was a significant increase in the activity of ADH III isoenzyme (15.06 mU/l and 14.62 mU/l vs. 11.82 mU/l; p<0.001) and total ADH activity (764 mU/l and 735 mU/l vs. 568 mU/l) in the sera of patients with acute pancreatitis or chronic pancreatitis compared to the control. The diagnostic sensitivity for ADH III was about 84%, specificity was 92 %, positive and negative predictive values were 93% and 87% respectively in acute pancreatitis. Area under the Receiver Operating Curve (ROC) curve for ADH III in AP and CP was 0.88 and 0.86 respectively. CONCLUSION: ADH III has a potential role as a marker of acute and chronic pancreatitis.

The alterations in alcohol dehydrogenase and aldehyde dehydrogenase activities in the sera of patients with renal cell carcinoma.

PURPOSE: In a previous study we showed that the total activity of alcohol dehydrogenase (ADH) and its isoenzyme class I was significantly higher in renal cancer (RCC) cells compared to normal kidney. The aim of this study was to compare the activities of ADH isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with different stages of RCC and healthy subjects. MATERIALS AND METHODS: Serum samples were taken from 54 patients with clear cell RCC (17 patients in stage II, 22 in stage III and 15 in stage IV) and 52 healthy patients. Class III, IV of ADH and the total ADH activity was measured by the photometric method. For the measurement of ADH class I, II and the total ALDH activity we employed the fluorometric method. RESULTS: The total activity of ADH and its isoenzyme class I were significantly higher in the sera of patients with every stage of RCC compared to healthy subjects. The analysis of ALDH activity did not indicate significant differences between tested groups. CONCLUSIONS: The increased activity of total ADH and its isoenzyme class I in the sera of patients with RCC, seems to be caused by isoenzymes being released from cancerous cells and may be useful for diagnostics of renal cancer.

The Diagnostic Significance of Serum Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase Activity in Urinary Bladder Cancer Patients.

BACKGROUND: The aim of this study was to investigate a potential role of alcohol dehydrogenase and aldehyde dehydrogenase as tumor markers for urinary bladder cancer. MATERIALS AND METHODS: Serum samples were obtained from 41 patients with bladder cancer and 52 healthy individuals. Class III and IV of ADH and total ADH activity were measured by the photometric method. For measurement of class I and II ADH and ALDH activity, the fluorometric method was employed. RESULTS: Significantly higher total activity of ADH was found in sera of both, low-grade and high-grade bladder cancer patients. The diagnostic sensitivity for total ADH activity was 81.5%, specificity 98.1%, positive (PPV) and negative (NPV) predictive values were 97.4% and 92.3% respectively. Area under ROC curve for total ADH activity was 0.848. CONCLUSION: A potential role of total ADH activity as a marker for bladder cancer, is herein proposed.

Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study.

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD+) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD+ and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD+, and pyruvate metabolized ethanol 20-40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD+ in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

The activity of class I, II, III and IV alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in the sera of bladder cancer patients.

OBJECTIVES: Studies on alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the sera of patients with malignant neoplasms show that cancer cells in many organs may release ADH isoenzymes into the blood. The aim of this study was to investigate the differences in the activity of ADH isoenzymes and ALDH in the sera of patients with bladder cancer (BCa), and with different grades of the disease. MATERIAL AND METHODS: Blood samples were taken from 39 patients with BCa (15 patients with low-grade and 24 with high-grade BCa) and from 60 healthy subjects. Class III and IV of ADH and total ADH activity were measured using the photometric method, while class I and II ADH and ALDH activity using the fluorometric method with class-specific fluorogenic substrates. RESULTS: The activity of the class I ADH isoenzyme and total ADH was significantly higher in the sera of BCa patients as compared to control group. Analysis of ALDH activity did not show statistically significant differences between the tested groups. Significantly higher total activity of ADH in comparison to control was found in both, low-grade and high-grade BCa group. The activity of ADH class I was also significantly higher in high-grade BCa group when compared to low-grade patients and controls. CONCLUSION: The increase of total ADH activity in the sera of BCa patients seems to be caused by isoenzymes released from cancerous cells. The higher activity of ADH I probably resulted from metastatic tumors as significant increase was detected only in the sera of high-grade bladder cancer patients.

Circulating Tumor Cells Expressing Markers of Tumor-Initiating Cells Predict Poor Survival and Cancer Recurrence in Patients with Pancreatic Ductal Adenocarcinoma.

Purpose: Circulating tumor cells (CTCs) have been identified in the blood of patients with pancreatic adenocarcinoma (PDAC), but little is known about the exact phenotype of these cells. We assessed expression of aldehyde dehydrogenase (ALDH), CD133, and CD44 as markers of CTCs with a tumor-initiating cell (TIC) phenotype in patients with PDAC and the relationship of this expression to patient outcomes.Experimental Design: Peripheral blood from 60 consecutive patients with PDAC undergoing surgical resection was obtained and processed using the Isolation by Size of Epithelial Tumor (ISET) method. Immunofluorescence was used to identify CTCs expressing cytokeratin, CD133, CD44, and ALDH.Results: Forty-seven patients (78%) had epithelial CTCs staining positive for pan-cytokeratin and at least one TIC marker. Forty-six patients (77%) had epithelial CTCs that labeled with antibodies to cytokeratin and ALDH. By separate analysis, 34 (57%) had cytokeratin-positive, CD133-positive, and CD44-positive (triple-positive) CTCs, whereas 40 (67%) had cytokeratin-positive, CD133-positive, CD44-negative CTCs. The remaining 13 patients did not have CTCs, as defined by cytokeratin expression. ALDH-positive CTCs and triple-positive CTCs were significantly associated with worse survival by univariate analysis, even when accounting for other significant prognostic factors (all, P ≤ 0.01). ALDH-positive CTCs, triple-positive CTCs, and dual cytokeratin- and CD133-positive CTCs were independent predictors of tumor recurrence by logistic regression analysis and associated with decreased disease-free survival (all, P ≤ 0.03).Conclusions: CTCs labeling with one or more markers of TICs are found in a majority of patients with PDAC and are independently predictive of decreased disease-free and overall survival. Clin Cancer Res; 23(11); 2681-90. ©2016 AACR.

Alcohol dehydrogenase and aldehyde dehydrogenase in malignant neoplasms.

According to International Agency for Research on Cancer, ethanol and acetaldehyde belong to group 1 of human carcinogens. The accurate mechanism by which alcohol consumption enhances carcinogenesis is still unexplained. Alcohol is oxidized primarily by alcohol dehydrogenase (ADH) to acetaldehyde, a substance capable of initiating carcinogenesis by forming adducts with proteins and DNA and causing mutations. Next, acetaldehyde is metabolized by aldehyde dehydrogenase (ALDH) to acetate. In tissues of many cancers, we can observe significantly higher activity of total alcohol dehydrogenase with any change in aldehyde dehydrogenase activity in comparison with healthy cells. Moreover, in malignant diseases of digestive system, significantly increased activity of ADH isoenzymes class I, III and IV was found. The gynecological, brain and renal cancers exhibit increased activity of class I ADH. ADH and ALDH can play also a crucial regulatory role in initiation and progression of malignant diseases by participation in retinoic acid synthesis and elimination of toxic acetaldehyde. Besides, changes of enzymes activities in tumor cells are reflected in serum of cancer patients, which create the possibilities of application ADH isoenzymes as cancer markers.

[The effect of subchronic inhalations of nitric oxide on metabolic processes in blood of experimental animals].

Metabolic processes were investigated in plasma and erythrocytes of Wistar rats exposed to daily 10-min sessions of NO inhalation for 30 days. These included determination of glucose and lactate, catalase activity, and activities of aldehyde dehydrogenase (ALDH), lactate dehydrogenase (LDH), and catalase. NO inhalation in a concentration of 20 ppm, 50 ppm and 100 ppm caused an increase in glucose and lactate. Inhalation of 100 ppm NO also increased catalase activity. Inhalation of all NO concentrations resulted in a decrease of ALDH activity, while the decrease in LDH activity was observed at NO concentrations of 50-100 ppm.

The Diagnostic Value of Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase Measurement Sera of Cervical Cancer Patients.

AIM: The aim of this study was to investigate a potential role of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) as tumor markers for cervical cancer. MATERIALS AND METHODS: Blood samples were obtained from 43 women with cervical cancer. Isoenzymes class III, IV of ADH and total ADH activity were measured in the sera by the photometric method and class I, II ADH and ALDH activity by the fluorometric method. RESULTS: The total activity of ADH and ADH I was significantly higher in the serum of patients with cervical cancer than in control groups. The diagnostic sensitivity for ADH I was 61,76%, specificity 65,7%, PPV and NPV were 70 and 62,16% respectively. AUC for ADH I was 0,654 and for total ADH 0,618. CONCLUSION: The results suggest a potential role of ADH I as a marker for cervical cancer.

The diagnostic significance of serum alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase activity in renal cell cancer patients.

OBJECTIVES: In previous experiments, we have found an increased level of class I ADH and total ADH activity in RCC tissues. Changes in cancer cells may be reflected by ADH activity in the serum and could thus be helpful for diagnostics of renal cancer. The aim of this study was to investigate a potential role of ADH and ALDH as tumor markers for RCC. MATERIAL AND METHODS: Serum samples were taken from 59 patients with RCC and 52 healthy subjects. Class III and IV of ADH and total ADH activity was measured by the photometric method. For measurement of class I and II ADH and ALDH activity, we employed the fluorometric method. RESULTS: The total activity of ADH and ADH I was significantly higher in the serum of patients with every stage of RCC compared to healthy subjects. The diagnostics criteria was higher for ADH I than for total ADH activity. The diagnostic sensitivity for ADH I was 73.36%, specificity 85.61%, predictive values of positive and negative results were 79.12 and 75.03% respectively. Area under ROC curve for ADH I was 0.748 and for total ADH 0.689. CONCLUSION: The results suggest a potential role of ADH I as a marker for RCC.

Serum Alcohol Dehydrogenase and Aldehyde Dehydrogenase Activity in the Course of Hepatitis C.

BACKGROUND: Hepatistis C virus (HCV) affects approximately 170 million people, and it is the leading cause of the chronic liver disease. The destruction of liver cells is reflected by an increase of different enzyme activities in the serum. These enzymes include alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which play a significant role in the metabolism of many biological substances and exist mainly in the liver. In this study we investigated the activity of alcohol dehydrogenase and its isoenzymes and the total activity of ALDH in the sera of patients with hepatitis C. METHODS: Serum samples were taken for routine biochemical investigations from 50 patients with hepatitis C and from 50 healthy subjects. The activity of class I and II ADH isoenzymes and ALDH activity were measured by spectrofluorometric methods. For the measurement of total ADH activity and activity of class III and IV isoenzymes, the photometric methods were used. RESULTS: The analysis of our results shows a statistically significant increase in the activity of ADH I and ADH II (2.5-fold and 2-fold, respectively). Activities of both classes of alcohol dehydrogenase isoenzymes have good correlation with alanine and aspartate aminotransferase. The observed increase in total alcohol dehydrogenase activity was not very high but confirmed the elevation of class I and II isoenzyme activity. CONCLUSIONS: We can state that the activity of class I and II alcohol dehydrogenase isoenzymes in the sera of patients with hepatitis C is increased and it seems to be caused by the release of these isoenzymes from damaged liver cells.

Exploring the heterogeneity of the hematopoietic stem and progenitor cell pool in cord blood: simultaneous staining for side population, aldehyde dehydrogenase activity, and CD34 expression.

BACKGROUND: The stem cell content in cord blood (CB) units is routinely assessed regarding nucleated cells, CD34+ cell count, and number of colony-forming units (CFUs). Efforts are made toward finding better ways of defining stemness of CB units. Side population (SP) phenotype and activity of aldehyde dehydrogenase (ALDH) are functional markers of stemness that can be assayed using flow cytometry. STUDY DESIGN AND METHODS: We have developed a protocol for simultaneous determination of CD34+, SP, and ALDH+ populations in relation to immature white blood cells (CD45dim) in CB. Viable nucleated cells were consecutively stained for SP and ALDH activity and with antibodies against the CD45, CD34, and CD117 antigens. RESULTS: The SP and ALDH+ populations could reliably be measured simultaneously. The median sizes of the SP and the ALDH+ populations were 0.85 and 3.3% of CD45dim cells, respectively. There was no overlap between the SP and ALDH+ populations. Cells that were ALDH+ expressed CD34 and CD117, but SP cells were negative for these markers. The ALDH+ cell content correlated with CD34+ cell content (p < 0.001) and with CFU-granulocyte-macrophage (GM; p = 0.03) but not with total CFUs. SP did not correlate with CD34+, CFU-GM, or total CFU. CONCLUSIONS: We show that simultaneous detection of the CD34, SP, and ALDH+ cells is clearly feasible using only small amounts of CB. In CB, ALDH+, and CD34+ cells are overlapping populations distinctly separated from the SP population. The difference in relation to the capacity for colony growth between ALDH+ and SP underlines that they define different cell populations.