James T. Wu

Serum alpha fetoprotein (AFP) levels in normal infants.

Summary: Serum α-fetoprotein (AFP) levels were monitored in 32 normal babies consecutively from 2 to 3 days, 2 wk, and 2 and 4 months after birth. In addition, serum AFP concentration was also measured in 116 random specimens from infants with normal liver enzymes and 10 infants born immaturely. Results were combined to establish normal AFP levels for infants at various ages. Serum AFP disappeared rapidly after birth. We found that it was not until 8 months of age that the normal AFP level in infants approached adult level. The half-lives of AFP degradation were estimated to be 5.5 days between birth and 2 wk, 11 days between 2 wk to 2 months, and 33 days between 2 and 4 months of age. In contrast to earlier belief, we felt that some AFP synthesis still exists after birth; however, the rate of synthesis may also decrease with age.Speculation: Our results favor the view that some α-fetoprotein (AFP) synthesis exists after birth. The rate of synthesis is slowly decreased with time. Any factor affecting the rate of synthesis may influence the serum AFP level. The wide range of variation in serum AFP levels in infants of various ages indicates that there must be more than one unknown factor affecting the AFP serum level. The normal range will be narrower if these factors become known. By using our age-dependent normal AFP serum levels, it would be interesting to see whether differential diagnosis of neonatal hepatitis from biliary atresia can be unproved and whether infants do have higher and more frequent elevation of serum AFP with hepatic disorders.

Cell-free DNA: measurement in various carcinomas and establishment of normal reference range.

BACKGROUND Cell-free DNA is detectable in circulating blood. Numerous reports in the literature have pointed out that cell-free DNA in plasma or serum has the clinical potential to be a more specific tumor marker for the diagnosis and prognosis, as well as the early detection, of cancer. METHODS In order to adapt cell-free DNA to a routine clinical laboratory test, we used commercial kits such as the QIAamp blood kit for DNA extraction and the PicoGreen DNA kit for DNA quantification. This was done so our results and the normal reference value established would allow to be compared by other laboratories. We have established the normal reference level of cell-free DNA for females and males from age 20-70 years. We also detected elevated cell-free DNA in all cancers that were tested in this study, including carcinomas, leukemia and lymphoma. RESULTS Our study indicates that the elevation of serum cell-free DNA was usually detected in specimens containing elevated tumor markers and is most likely associated with tumor metastases. The electrophoretic pattern of cell-free DNA showed that cell-free DNA from cancer patient is fragmented, containing smaller DNA (100 bp) not found in normal cell-free DNA. CONCLUSIONS Measuring cell-free DNA may complement currently used tumor markers for the management of cancer patients.

Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers

BACKGROUND 8-hydroxydeoxyguanosine (8-OHdG) is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Urinary 8-OHdG is now considered as a biomarker of generalized, cellular oxidative stress and is linked to degenerative diseases including cancer. METHODS We developed a competitive enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG by coating BSA conjugated 8-hydroxyguanine (8-OHG) on a microplate. Urine specimens containing 8-OHdG and monoclonal anti-8-OHdG antibody were incubated together in the microwell. Final quantification of bound anti-8-OHdG antibody was estimated by the addition of HRP-conjugated sheep-anti-mouse antibody. RESULTS The concentration range of the calibration curve was 0-60 ng/ml. The sensitivity of the assay was 0.5 ng/ml. The within-day precision and day-to-day precision were <10%. The ELISA correlated well with a commercial kit (r=0.9). Our assay measured not only 8-OHdG but also 8-OHG and 8-hyroxyguanine in urine. Increased urinary concentration of 8-OHdG and its analogs were detected in both patients with bladder cancer (70.5+/-38.2 ng/mg creatinine) and prostate cancer (58.8+/-43.4 ng/mg creatinine) as compared to the healthy control (36.1+/-24.5 ng/mg creatinine). CONCLUSION Our preliminary data suggest that the competitive ELISA for 8-OHdG and its analogs appears to be a simple method for quantifying the extent of oxidative stress and may have potential for identifying cancer risk.

Elevated cell-free serum DNA detected in patients with myocardial infarction

BACKGROUND Cell-free DNA is detectable in the circulation. Increased cell-free DNA has been detected in cancer patients and individuals with trauma. We want to know whether patients with myocardial infarction (MI) also have increased cell-free DNA in their blood. METHODS We used a QIAamp blood kit for DNA extraction from serum and the PicoGreen DNA kit for quantification. DNA patterns of serum DNA were established by gel electrophoresis on 2.5% metarphor gel. RESULTS The average serum DNA in MI patients (N=55) was 511+/-398 ng/ml, more than 10-fold higher than normal (36.3+/-23.8 ng/ml, n=274). Patients with increased CK-MB (>4%) were associated with highly increased concentrations of cell-free DNA (93.4%). There was no correlation between the concentration of cell-free DNA and the concentrations of CK-MB, troponin I and C-reactive protein. In serial specimens, we found that the cell-free DNA rose early, but peaked behind CK-MB. A slightly diffused DNA ladder could be found with pooled cell-free DNA from MI patients by electrophoresis with the smallest DNA band at only a few hundred base pairs. CONCLUSIONS Cell-free DNA in MI patients is increased in patients diagnosed with MI, and may complement troponin and CK-MB in a multiple marker test format.

Hyperhomocysteinemia is a risk factor for cancer and a new potential tumor marker

Plasma homocysteine (Hcy), a well-known independent risk factor for coronary heart disease, is also a risk factor for cancer. Results from our studies indicate that Hcy could be used as a tumor marker. We found elevated circulating total homocysteine (tHcy) in cancer patients even though they were not treated with anti-folate drugs. In serial specimens from cancer patients undergoing treatment, the change of tHcy coincided with the concentration of tumor markers. The rapid proliferation of tumor cells contributed to the much higher concentrations of circulating tHcy. Both concentrations of tHcy and tumor marker would increase in parallel during the growth of tumor cell, but only the Hcy concentration would decline in response to tumor cell death. Several biochemical changes, including folate deficiency, oxidative stress, aberrant DNA methylation, and production of homocysteine thiolactone have been identified in association with hyperhomocysteinemia, which explained why elevated homocysteine eventually led to carcinogenesis. Conceivably, tHcy may be used as a more accurate tumor marker for monitoring cancer patients during treatment, and hyperhomocysteinemia as a risk factor for carcinogenesis.

Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range

BACKGROUND C-reactive protein (CRP), a marker of systemic inflammation, has been proposed to predict outcome in patients with unstable angina; and elevated levels of CRP were found to be associated with an increased risk of coronary events. METHODS Two enzyme-linked immunosorbent assays (ELISA) of different sensitivities were developed on microplate for CRP. Both ELISA established used Dako polyclonal anti-CRP antibody for coating and Dako horse radish peroxidase (HRP)-conjugated polyclonal anti-CRP antibody for detection. RESULTS The sensitivity of the high and regular sensitivity ELISA was 0.16 and 0.6 mg/l, respectively. Our assays demonstrated an excellent correlation with commercial CRP assays performed on a Behring Nephelometer Analyzer II (BNII) at both regular and ultrasensitive levels, with both correlation coefficients above 0.98 and slopes of approximately 1. Using our microplate assays, we established normal reference value for serum CRP. Based on ANOVA statistical test, we found that the mean +/- S.D. was 1.3 +/- 1.27 mg/l (n=202) for normal individuals of 50-80 years and 0.43 +/- 0.42 mg/l (n=148) for the group of 20-50 years. CONCLUSIONS The normal serum CRP mean concentrations for two age groups were distinctively different (p value<0.001). Our study suggests two different normal cutoffs of serum CRP to be employed for individuals in different age groups.

Serum total homocysteine increases with the rapid proliferation rate of tumor cells and decline upon cell death: a potential new tumor marker

BACKGROUND We were interested to know why cancer patients are frequently associated with elevated circulating total homocysteine (tHcy) even though they are not treated with anti-folate drugs. METHODS We employed tissue cultures to compare both the homocysteine (Hcy)-released and production of tumor markers between tumor and normal cell lines. RESULTS We detected much higher concentrations of homocysteine (Hcy) released by the tumor cells. However, much less difference was found between normal and tumor cell lines when Hcy concentration was expressed per the same number of cells. During the cell culture, the increase of Hcy and the increase of tumor marker concentration paralleled each other for the first 7 days. After the seventh day of the culture when cells started dying, tumor markers continued to rise, whereas levels of Hcy and cell numbers leveled off. We found that the serum concentration of Hcy fluctuated in circulation coinciding with that of tumor marker in individual cancer patients unless taking anti-neoplastic drug. CONCLUSIONS The elevation of tHcy concentration may be caused by the rapid tumor cell proliferation and reflect only the number of live cells. Serum Hcy may be a potentially useful tumor marker to monitor tumor activity.

Serum chromogranin A: early detection of hormonal resistance in prostate cancer patients.

We monitored both chromogranin A (CgA) and neuron specific enolase (NSE) in serial serum specimens from 14 patients with prostate cancer (CAP patients) showing resistance to hormonal treatment. Elevated serum CgA was detected in 10 out of these 14 patients (71%) during treatment, and an early appearance of elevated serum CgA was found in 6 of 14 (43%) of these patients when serum tPSA levels were still in the normal range. If patients with radical prostatectomy were not included, the percentage of patients showing an early appearance of elevated serum CgA would have been much higher. Elevated serum CgA levels also were found in patients not subject to hormonal therapy. Serial specimens from two out of three prostate cancer patients, randomly selected, contained elevated serum CgA. Serum NSE was not detectable in any of the serial specimens we studied, suggesting that CgA, not NSE, should be used as a marker for neuroendocrine differentiation. We also compared the serum CgA in random serum specimens between patients with BPH (benign prostate hyperplasia) and with prostate cancer in the concentration range of serum tPSA between 3–15 ng/mL. Although serum CgA concentrations in BPH patients overlapped considerably with those levels in patients with prostate cancer, levels > 100 ng/mL should suggest prostate cancer. The early appearance of elevated serum CgA allows an early change of therapy to be made and can lead to the effective prevention of any further development of metastases. J. Clin. Lab. Anal. 12:20–25, 1998.

Advanced glycation end product (AGE): Characterization of the products from the reaction between D-glucose and serum albumin

We incubated bovine serum albumin (BSA) with glucose in an attempt to study how the advanced glycation end products (AGEs) are formed and what methods can be used for their identification and isolation. The reaction was monitored by boronated affinity gel, size exclusion and ion exchange chromatography, and chromatofocusing. Reaction products were also characterized by fluorescence (excitation, 370 nm; emission, 440 nm) we found that the AGEs could be detected as early as after 3 days incubation. The fluorescence was always associated with the larger molecules of cross‐linking product resulting from the reaction between BSA and glucose. The overall fluorescence intensity increased with incubation time and fluorescence of the highest intensity was found with the AGE product largest in size. As with the Amadori product, AGEs also bind to the boronated gel column but with an even higher afflinity. Compared to the original albumin monomer AGE molecules are not only larger in size but also have lower isoelectric points and carry more negative charges. Both the size and the negative charges of AGEs continue to increase over time during incubation. This results in a group of cross‐linking molecules heterogeneous in size and charge. These results will aid in both the isolation and selection of appropriate AGE molecules for the preparation of anti‐AGE antibodies, calibrator, and control in the development of an AGE immunoassay.

Advantages of replacing the total PSA assay with the assay for PSA-α1-antichymotrypsin complex for the screening and management of prostate cancer

Several advantages become immediately apparent when the prostate specific antigen (PSA, or tPSA) assay is replaced by the assay specific for the serum PSA‐α1‐antichymotrypsin (PSA‐ACT) complex. For instance, random contributions to the tPSA value by various serum minor PSA isoforms can be avoided, making possible the determination of a more accurate relation of the PSA‐ACT concentration to the tumor activity. Discrepancies in percent free PSA (% fPSA) values from the same specimens due to the use of different commercial kits also can be eliminated, mainly because the PSA‐ACT assay does not have the problems in antibody selection and calibrator preparation usually associated with the tPSA assay. We found that at the present time different cutoffs of % fPSA for the differentiation of BPH from prostate cancer must be established for each individual tPSA assay. Cutoffs established using values from one tPSA assay should not be used for making clinical decisions when their tPSA values are determined by a different kit. Moreover, when we monitored the patients during treatment with serum tPSA, specific fPSA, and specific PSA‐ACT complex assays simultaneously, it was clear that any interpretation of the patients clinical status based on tPSA values alone could be misleading. Because there is less PSA‐ACT complex in BPH specimens relative to that found in cancer serum samples, expressing fPSA as “fPSA/PSA‐ACT × 100” and measuring PSA‐ACT complex concentrations instead of tPSA during screening improve the measurable contrast between BPH and prostate cancer. Although individually modest, collectively these advantages can add up to considerable improvements. J. Clin. Lab. Anal. 12:32–40, 1998.