Study of heavy metal poisoning in frequent users of Chinese medicines in Hong Kong and Macau.

This study is a direct assessment of blood heavy metal concentrations of frequent users of Chinese medicines (CM), who had been taking prescribed CM at least 6 days per week for not less than 3 months, to determine whether their intake of CM could cause an increased load of toxic heavy metals in the body. From November 2009 to June 2010, 85 subjects were recruited with informed consent, and their blood samples were collected for measurement of arsenic, cadmium, lead and mercury concentrations. Results showed that blood concentrations of four heavy metals of nearly all 85 subjects were within reference ranges. Only one subject who had consumed plentiful seafood was found to have transiently increased blood arsenic concentration (29% higher than the upper limit of the reference range). However, after refraining from eating seafood for 1 month, his blood arsenic concentration returned to normal. Eighty commonly prescribed CM in both raw medicine and powder concentrate supplied by local distributors were also tested for the four heavy metals. Twelve out of the 80 raw medicines were found to contain one or more of the heavy metals that exceeded the respective maximum permitted content. Cadmium was most frequently found in the contaminated samples. None of the powder concentrates had heavy metal content exceeding their respective maximum permitted level.

Serum small-dense LDL abnormalities in chronic renal disease patients.

Cardiovascular disease (CVD) is the principal cause of mortality in chronic kidney disease (CKD) patients. Dyslipoproteinaemia is a common metabolic derangement in CKD and a traditional risk factor for CVD. This study investigates serum lipoprotein, especially small-dense low-density lipoprotein (sd-LDL), abnormalities in CKD patients. A total of 131 CKD patients (age: 59 +/- 12 years, male = 64) diagnosed according to Kidney Disease: Improving Global Outcomes, 2004 (KDIGO) and 121 age- and gender-matched control subjects (age: 58 +/- 6 years, male = 62) were recruited from Hong Kong and Macau. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C) and direct LDL-C were assayed enzymatically. In addition, sd-LDL, together with very low density and intermediate-density lipoproteins (VLDL and IDL) were measured by US Food and Drug Administration (FDA)-approved polyacrylamide gradient gel electrophoresis. Compared to controls, CKD patients showed significantly decreased TC, LDL-C, normal-size LDL and HDL-C with increased TG, VLDL, IDL and sd-LDL (all P < 0.01). The increased sd-LDL and decreased normal-size LDL fractions resulted in a significantly elevated sd-LDL:LDL ratio in CKD (P < 0.005). In contrast to the low TC and LDL-C, sd-LDL and sd-LDL:LDL ratio were significantly elevated in CKD. Thus, sd-LDL will be used increasingly for CVD risk assessment in CKD and other diseases that show lipoprotein derangement.

Human papillomavirus infection in Macau women.

OBJECTIVE: To investigate the prevalence and genotypes of human papillomavirus (HPV) infection in Macau women. STUDY DESIGN: Female patients presenting for a medical consultation or medical check-up were recruited with informed consent. METHODS: Cytology and HPV-DNA genotyping were performed on 402 cervical specimens that were collected from Macau women. RESULTS: Of the specimens, 29.9% were found to be HPV-DNA positive; 26.4% were infected with one HPV genotype, while 3.0% and 0.5% were infected with two and three HPV genotypes, respectively. The most prevalent HPV genotype was type 52 (11.1%), followed by type 16 (9.7%). Both types 51 and 62 ranked third (9.0%). CONCLUSIONS: The HPV infection rate in Macau appears to be higher than that in the neighbouring city of Hong Kong. The most prevalent genotypes were similar to those in South-west and Southern China.

Prevalence of hepatitis B genotype and viral basic core promoter and precore mutations among teenagers in Macao: relationship with hepatocellular carcinoma development.

Chronic hepatitis B virus (HBV) infection is a global problem and over 75% of cases are reported in the Asia Pacific region. Infection can lead to progressive liver disease, cirrhosis and hepatocellular carcinoma (HCC). Previous studies suggest the prevalence of HBV carriers in Macau to be approximately 10% of the population. This study aims to investigate the prevalence of HBV genotypes among HBV-positive teenagers in Macao and the prevalence of base core promoter (BCP) and precore (PreC) mutations in the viral genome. In addition, through monitoring aminotransferase and alpha-fetoprotein, it aims to investigate relationships among HBV genotypes, BCP/PreC mutations and HCC development. This study recruited 1991 teenagers in Macau in 2008, and the PreS1/S2, BCP and PreC region of the HBV genome from 34 HBsAg-positive subjects were amplified and sequenced to determine HBV genotype and presence of HCC-associated mutations. Results suggested that the average rate of HBV infection among secondary school teenagers in Macao is low, and HBV genotype B and C viruses were found to predominate in Macao. The BCP/PreC mutations A1762T, G1764A, G1896A and C1766T were identified in 2.9-11.7% of subjects. However, no significant relationship was observed between HBV genotype, BCP/PreC mutations and HCC development.

Chemical pattern-aided classification to simplify the intricacy of morphological taxonomy of Epimedium species using chromatographic fingerprinting.

Epimedium herb (Yinyanghuo), one of the popular Chinese materia medica, is a multiple species colony of Epimedium genus belonging to Berberidaceae. There are five species of Epimedium that have been officially adopted in Chinese Pharmacopoeia under the same crude drug name 'Yinyanghuo' comprising Epimedium brevicornu, E. koreanum, E. sagittatum, E. pubescens, and E. wushanense. In addition, non-official species like E. acuminatum, E. miryanthum and E. leptorrhizum are also mix-used. Frequently, the morphological taxonomical identification is very difficult during on-site inspection for species authentication in the market. Researchers are often bewildered by the multiple species ambiguity when putting this crude drug in use. Referring to the bioactive constituents that are vital for therapeutic efficacy, the key to clarifying the multiple species confusion should rely on analysis of the bioactive composition. It is well known that medicinal Epimedium herbs contain special C-8 prenylated flavonol glycosides which contribute to various bioactivities and the major four, epimedin A (A), epimedin B (B), epimedin C (C) and icariin (I), are unanimously used as bioactive markers for quality control. In this study, HPLC-DAD fingerprinting was performed for investigating the molecular spectrum of various Epimedium species. It was found that the four major flavonoids constitute the middle part of the chromatographic profiles to form a specific region (named as 'ABCI fingerprint region') being dominant in the HPLC profiles of all medicinal Epimedium species, and the five official species express five different 'ABCI' patterns (different peak: peak ratios). Our study found that the convergent tendency of the 'ABCI region' among multiple species of Epimedium could facilitate differentiation of complex commercial samples based on similar bioactive composition should confer similar bioactivities. Merging the different species that possess the same 'ABCI region' pattern into the same group can create a simpler bioactive-fraction-aided classification array by clustering the commercial samples into three bioactive ingredients-based fingerprint patterns - 'E.b. pattern', 'E.k. pattern' and 'extensive E.w. pattern'. This approach offers the feasibility of characterizing and quality-controlling complex samples in the same genus designated under a single herbal drug entity on the premise of possessing the same bioactive ingredients pattern and supported by long-term traditional usage.

The effects of a Chinese medicinal suppository (Vitalliver) on insulin-like growth factor 1 and homocysteine in patients with hepatitis B infection.

The liver is the major organ for the metabolism of homocysteine (Hcy) and production of insulin-like growth factor 1 (IGF-1). Hcy metabolism and IGF-1 synthesis may be impaired in chronic liver diseases. The study investigated the regulatory effect of a Chinese herbal suppository, Vitalliver, on Hcy and IGF-1, as well as their relationship in patients with hepatitis B infection. Forty patients with chronic hepatitis B virus (HBV) infection without cirrhosis, 25 males and 15 females, were observed for changes in Hcy and IGF-1 after the administration of Vitalliver (one nightly) for a period of 3 months. Serum levels of Hcy, IGF-1 and IGFBP-3 were measured at baseline, and at 1 month and 3 months after treatment. Vitalliver reduced Hcy levels significantly (p = 0.001) from 9.7 +/- 2.8 to 9.0 +/- 2.1 micromol/L after treatment of 3 months. Furthermore, the IGF-1 levels increased significantly (p < 0.001) from 170.2 +/- 81.8 to 212.8 +/- 80.9 ng/mL at 1 month and 187.5 +/- 72.3 ng/mL at 3 months (p = 0.001) after treatment. In conclusion, it is speculated Vitalliver may have a self-regulatory effect on the release of IGF-1 in HBV patients without liver cirrhosis.

Protective effects of Danshensu from the aqueous extract of Salvia miltiorrhiza (Danshen) against homocysteine-induced endothelial dysfunction.

Homocysteine (Hcy) is a by-product of methionine metabolism. An imbalance of Hcy in the body may lead to hyperhomocysteinemia, a condition with elevated Hcy concentration in blood that may be one of the risk factors responsible for the development of several vascular diseases (thromboembolism, atherosclerosis, stroke, vascular diseases and dementia). Radix Salvia miltiorrhiza (Danshen), a well-known Chinese medicinal herb that can activate and improve blood microcirculation, is noticeable for its beneficial effect in treating cardiovascular diseases. The present study is to demonstrate the protective effect of Danshen extract against the homocysteine-induced adverse effect on human umbilical vein endothelial cell (HUVEC). Homocysteine (5 mM) not only decreased the cell viability but also caused the disruption of capillary-like structure formation in vitro. The protective effect of Danshen aqueous extract and its active compounds on endothelial cell function were demonstrated through an in vitro tube formation assay, which mimics the new blood vessel formation. To identify the active components in the aqueous extract of Danshen, the content was characterized by instrumental analysis using high performance liquid chromatography with diode array detector (DAD) and electrospray tandem mass spectrometry (ESI-MS/MS). Interestingly, Danshen extract and its pure compounds showed different effectiveness in protecting HUVEC against Hcy-induced injury according to the following descending order: Danshen aqueous extract, 3-(3,4-dihydroxy-phenyl)-2-hydroxy-propionic acid (Danshensu), protocatechuic acid, catechin and protocatechualdehyde. We believed that such findings might provide evidence in understanding the beneficial effects of Danshen on the cardiovascular system.

A panel study on the effects of a Chinese medicinal suppository, Vigconic VI-28, on insulin-like growth factor 1 and homocysteine in healthy men.

Changes in serum homocysteine (Hcy), often related to stroke and vascular dementia, are negatively correlated with changes in serum insulin-like growth factor 1 (IGF-1) and growth hormone (GH) replacement decreases Hcy levels in men with GH deficiency. Very little information on the effects of Chinese medicines on GH and Hcy is available in the literature published in English. In this study, the effects of a Chinese medicine suppository, Vigconic VI-28 (VI-28), consisting of concentrated extracts of a composite mixture of herbal materials, on serum IGF-1 and Hcy were studied. In vivo observations after treatment with Chinese medicines have often indicated changes in biochemical profiles of measurable parameters related to those changes in endocrine secretions. Thirty six healthy males (age 47-66) were under observation over a 16-week schedule after using VI-28 suppository from 0 to 12 weeks. Blood specimens were taken monthly (except at the end of week 8) for analysis of Hcy and IGF-1 levels. Compared with week 0, IGF-1 levels (192.5 +/- 66.4 ng/ml) were significantly elevated at week 4 (211.7 +/- 80.5, p < 0.05) and week 12 (226.6 +/- 95.2 ng/ml, p = 0.01). No significant changes were observed for Hcy for the whole cohort from week 0 to week 16. When the cohort was divided into 2 groups using a Hcy level of 13.0 micro mol/l as the cut-off, a significant (p < 0.05) difference in IGF-1 was observed between the 2 groups at week 12 only. The mean IGF-1 of 14 subjects with higher Hcy levels was lower than that of the 22 subjects with lower Hcy. We believe that VI-28 may exert a regulatory effect on the relationship between Hcy and IGF-1, at least in subjects with relatively low levels of Hcy. In addition, we also observed an apparent association of hyperhomocysteinemia (Hcy> or =13.0 micromol/l) with decreased IGF-1.

Charge distribution of IgA-lambda in IgA nephropathy.

The finding that eluted mesangial IgA and serum IgA from patients with IgA nephropathy had a restricted anionic charge contrasting with normal serum IgA prompted us to study the charge of the kappa and lambda subclasses of IgA. Serum IgA from 11 patients with IgA nephropathy and 11 controls was purified by affinity chromatography by binding to jacalin. The charges of IgA were studied by a novel method. The spectrotype of total IgA was first studied by isoelectric focusing and immunoblotting. IgA lambda and IgA kappa was further analyzed by reacting with specific monoclonal antibodies. The amount of IgA with different pIs was analyzed by computerized densitometry. The anionic:cationic (A:C) ratio of IgA using pI 5.6 as the dividing point was greater in patients (at clinical quiescence and during exacerbation) than in controls (1.67 +/- 0.31 versus 1.36 +/- 0.27, p < 0.025). IgA lambda in both groups was anionic (A:C ratio 2.22 +/- 0.77 versus 2.36 +/- 0.36) and IgA kappa was cationic (A:C ratio 1.15 +/- 0.36 versus 1.04 +/- 0.39) but no difference in the A:C ratio was demonstrated. The increased A:C ratio in total IgA in patients was due to raised serum IgA lambda (kappa/lambda ratio 1.11 +/- 0.14 in patients and 1.51 +/- 0.16 in controls, p < 0.01). We had previously shown a predominant mesangial deposition of IgA lambda in IgA nephropathy. Animal experiments have revealed the preferential mesangial deposition of immune complexes is related to their anionic charges. Our data of raised anionic IgA lambda in IgA nephropathy may be important in determining its selective mesangial binding that could contribute to the immunopathogenesis.

Analysis of charge of kappa and lambda chain-containing serum immunoglobulin A by immunoblotting and densitometry.

A novel method for studying the charge distribution of different subclasses of serum immunoglobulin A (defined by the light chains) is described. Affinity-chromatography purified immunoglobulins were focused in polyacrylamide gel and were then transferred electrophoretically onto nitrocellulose membranes. The transferred immunoglobulins were detected by rabbit antiserum to human kappa (kappa) or lambda (lambda) chain, swine antirabbit IgG, rabbit anti-peroxidase antibodies and peroxidase, together with a substrate solution comprising H2O2 and diaminobenzidine. Finally, the developed membranes were made transparent with Triton-x 114, scanned at 485 nm with a densitometer to obtain quantitation of charge distribution.

Light-chain ratio of serum IgA1 in IgA nephropathy.

Patients with primary IgA nephropathy have deposits of IgA1 in their kidneys and increased IgA1 in circulation. We had previously shown that IgA nephritic patients displayed a unique immunological response characterized by a predominance of IgA with lambda chain in glomerular deposits and in circulation. We have now studied the kappa/lambda (kappa/lambda) ratio of serum IgA1 in 21 IgA nephritic patients at quiescence, with 11 patients investigated during exacerbation as well. A novel enzyme-linked immunosorbent assay was used with monoclonal mouse anti-human IgA1 as the solid-phase capture antibody and peroxidase-labeled anti-human kappa and lambda antibodies as tracers. The ratio of serum IgA1 to total IgA (mean +/- SD) was significantly higher in patients (90.1 +/- 8.2% at quiescence, P less than 0.01; 88.7 +/- 8.1% during exacerbation, P less than 0.02) than in 20 healthy age- and sex-matched controls (80.0 +/- 9.8%). Furthermore, serum IgA1 kappa/lambda ratios were significantly lower in patients (1.02 +/- 0.27 at quiescence, P less than 0.01; 0.93 +/- 0.16 during exacerbation, P less than 0.01) than in controls (1.31 +/- 0.30). These findings indicate a predominance of lambda light-chain IgA1 in the serum of IgA nephritic patients. However, no difference in IgA1 kappa/lambda ratio was observed in these patients at quiescence and during exacerbation.

Light chain ratios of serum immunoglobulins in disease.

We have determined the individual kappa (kappa)/lambda (lambda) ratios of serum IgG, IgA, and IgM in normal subjects and patients with rheumatoid arthritis, systemic lupus erythematosus, hepatic cirrhosis and IgA nephropathy--40 in each group. Serum samples were first screened by agarose electrophoresis to exclude paraproteinaemia. Concentrations of IgG, IgA, and IgM were determined by enzyme-linked immunosorbent assay (ELISA). The kappa and lambda chain concentrations of each immunoglobulin class were assayed by an ELISA method first developed by us for the determination of kappa/lambda ratios. Our results showed that kappa/lambda ratios of serum IgA and IgM were significantly different from that of IgG in normal subjects and the 4 groups of patients studied (p less than 0.01). The kappa/lambda ratios of individual immunoglobulins in patients with rheumatoid arthritis, systemic lupus erythematosus and liver cirrhosis were similar to those of normal subjects. However, patients with IgA nephropathy displayed a distinctly lower IgA kappa/lambda ratio, suggesting a unique antibody response in the immunopathogenesis of this disease.

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin.

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.

Light-chain ratios of immunoglobulins G, A, and M determined by enzyme immunoassay.

We describe an enzyme-linked immunosorbent assay for determination of light-chain ratios for IgG, IgA, and IgM in serum. A commercial serum with known overall kappa and lambda concentrations was used as standard. To capture the respective immunoglobulins, we used antibodies to gamma, lambda and mu chains coated onto microtiter plates. Peroxidase-conjugated anti-kappa and anti-lambda chain antisera were reacted with light chains on the captured immunoglobulins, and the amount of enzyme bound was monitored with o-phenylenediamine and urea-hydrogen peroxide as substrates. Calculation of absorbance ratios allowed determination of kappa and lambda chain concentrations of individual immunoglobulins in the standard and samples. Within-run and between-run CVs (n = 25) ranged from 5.9% to 13.0% for "high," "normal," and "low" kappa/lambda ratios for IgG, IgA, and IgM. The thoroughness of light-chain detection, expressed as, e.g., (IgA kappa + IgA lambda)/(total IgA), for 150 sera was 91-110%. The detection limit was 1 microgram/L. Reference intervals (mean +/- SD) for kappa/lambda ratios in sera from 100 apparently healthy adults were 2.34 +/- 0.80 for IgG, 1.59 +/- 0.40 for IgA, and 1.86 +/- 0.76 for IgM.

Enzyme-linked immunosorbent assay for determination of antibodies to xanthine oxidase.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect IgG, IgA and IgM antibodies to xanthine oxidase. The method used xanthine oxidase to coat sample wells on microtitre plates. The anti-xanthine oxidase concentrations were determined by reference to standard curves constructed by coating plates with anti-IgG, anti-IgA and anti-IgM to capture antibodies of different classes in standard human serum. The standard curves for IgG, IgA and IgM had a working range of 0 to about 60 ng/ml, and all results with commercial quality control serum fell within expected ranges. The coefficients of variation (CV) for within-batch precision (n = 30) and between-batch precision (n = 20) for IgG and IgM were about 9% and 12% respectively. The detection limit was 2 ng/ml. The ELISA was applied to assay serum samples of 110 Chinese and 110 European healthy subjects. A positively-skewed distribution in their anti-xanthine oxidase IgG and IgM levels was observed.

Enzyme-linked immunosorbent assay for alpha-fetoprotein in human serum.

A simple and inexpensive enzyme-linked immunosorbent assay for determination of serum alpha-fetoprotein concentration, with results available within 4 h, is described. The assay has a working range of 2-100 ng/ml, with within- and between-batch coefficients of variation (n = 24) at different concentrations between 8.9 and 12.7%. Results on 80 patients' sera (AFP concentrations 0-175 ng/ml) correlate well with those by radioimmunoassay. Turn around time (4 h) is shorter than with similar immunoassays currently available.

Enzyme-linked immunosorbent assay for free thyroxin in human serum.

We describe a simple enzyme-linked immunosorbent assay (ELISA) in which microtiter plates are used for determining the free thyroxin concentration (FT4) in serum. Only commercially available chemicals and reagents are needed, including the antiserum. The working range is 1 to 60 ng/L. Turnaround time is about 4 h. Within-run coefficients of variation (CV) for FT4 concentrations of 9.0, 19.0, and 35.0 ng/L were less than 5%; between-run CVs were 10% to 11%. Results by a radioimmunoassay method for 150 sera correlated well (r = 0.922).

Effect of ciclosporin on lymphocyte subpopulations and immunoglobulin production in IgA nephropathy.

The effect of ciclosporin on the cellular immunoregulation was examined in 19 patients with IgA nephropathy. The patients were randomly divided into two groups: 9 patients receiving oral ciclosporin (5 mg/kg/day) for 12 weeks and 10 patients receiving placebo and acting as controls. T lymphocyte subpopulations were determined using OKT monoclonal antibodies. The functional capability of lymphocytes was assessed by in vitro immunoglobulin synthesis of cultured peripheral mononuclear cells, thymidine uptake by cultured lymphocytes, and t lymphocyte activation with expression of interleukin-2 receptors. A fall of in vitro IgA production by cultured lymphocytes (p less than 0.05), a reduction of thymidine uptake by Staphylococcus aureus Cowan-stimulated cultured lymphocytes (p less than 0.05), and a reduction of activated lymphocytes expressing interleukin-2 receptor (p less than 0.05) were observed in patients after 12 weeks of ciclosporin therapy. The percentages of OKT4 and OKT8 lymphocytes and OKT4/8 ratios were not altered with therapy. A simultaneous reduction of proteinuria and a transient impairment of renal function were observed. Similar changes in cellular immune parameters and clinical response were not observed in the controls. Our study suggests ciclosporin could modulate the cellular immunity in IgA nephropathy.