The usefulness of the spontaneously hypertensive rat to model attention-deficit/hyperactivity disorder (ADHD) may be explained by the differential expression of dopamine-related genes in the brain.

Spontaneously hypertensive rats (SHR) are considered to represent a genetic animal model for attention-deficit hyperactivity disorder (ADHD). In the present studies, we compared the locomotor activity, learning and memory functions of juvenile male SHR, with age- and gender-matched genetic control Wistar-Kyoto rats (WKY). In addition, we investigated potential differences in brain morphology by magnetic resonance imaging (MRI). In other complimentary studies of the central nervous system, we used real-time PCR to examine the levels of several dopaminergic-related genes, including those coding for the five major subtypes of dopamine receptor (D1, D2, D3, D4 and D5), those coding for enzymes responsible for synthesizing tyrosine hydroxylase and dopamine-beta-hydroxylase, and those coding for the dopamine transporter. Our data revealed that SHR were more active than WKY in the open field (OF) test. Also, SHR appeared less attentive, exhibiting inhibition deficit, but in the absence of memory deficits relative to spatial learning. The MRI studies revealed that SHR had a significantly smaller vermis cerebelli and caudate-putamen (CPu), and there was also a significantly lower level of dopamine D4 receptor gene expression and protein synthesis in the prefrontal cortex (PFC) of SHR. However, there were no significant differences between the expression of other dopaminergic-related genes in the midbrain, prefrontal cortex, temporal cortex, striatum, or amygdala of SHR and WKY. The data are similar to the situation seen in ADHD patients, relative to normal volunteers, and it is possible that the hypo-dopaminergic state involves a down regulation of dopamine D4 receptors, rather than a general down-regulation of catecholamine synthesis. In conclusion, the molecular and behavioural data that we obtained provide new information that may be relevant to understanding ADHD in man.

Changes in RANKL/OPG/RANK gene expression in peripheral mononuclear cells following treatment with estrogen or raloxifene.

The RANKL/OPG/RANK pathway is the key mediator of osteoclastogenesis. Mononuclear cells may be implicated in post-menopausal osteoporosis. The effect of estrogen or raloxifene on bone resorption and the expression of RANKL/OPG/RANK in peripheral blood mononuclear cells (PBMCs) was examined. Twenty-nine women with post-menopausal osteoporosis were treated with estrogen (HRT) or raloxifene for 12 months. Bone mineral density (BMD) was measured at baseline and at 12 months at the spine and hip. Serum C-terminal telopeptide (CTX) and OPG were measured at baseline and at 1, 3, 6 and 12 months. PBMCs were isolated from 17 women and changes in RANKL, OPG and RANK mRNA were determined. The effects of estrogen or raloxifene in PBMCs in vitro were also assessed. BMD increased following treatment (lumbar spine % change mean [S.E.M.]: 4.3% [0.9], p<0.001). Serum CTX decreased (6 months: -43.7% [6.0], p<0.0001). Serum OPG declined gradually (12 months: -26.4% [4.4], p<0.001). RANKL, OPG and RANK gene expression decreased (6 months: RANKL 50.0% [24.8] p<0.001, OPG: 21.7% [28] p<0.001, RANK: 76.6% [10.2] p=0.015). Changes in OPG mRNA correlated with changes in BMD (r=-0.53, p=0.027) and CTX (r=0.7, p=0.0044). Down-regulation in RANKL, OPG, RANK mRNA and reduction in bone resorption was also seen in vitro. These results suggest that the expression of RANKL/OPG/RANK in PBMCs are responsive to the slowing in bone turnover/remodeling associated with treatment with estrogen or raloxifene. Further confirmatory studies are needed.

Polymorphisms in the P450 c17 (17-hydroxylase/17,20-Lyase) and P450 c19 (aromatase) genes: association with serum sex steroid concentrations and bone mineral density in postmenopausal women.

The CYP 17 and CYP 19 genes encode 17alpha-hydroxylase/17,20-lyase and aromatase, respectively, both involved in sex hormone synthesis. We investigated the association between 2 common polymorphisms in 1) the promoter region (T-->C substitution) of CYP 17, and 2) exon 3 (G-->A) of CYP 19, bone mineral density (BMD) and serum androgen/estradiol, in a case-control study of 252 postmenopausal women aged 64.5 +/- 9.2 yr (mean +/- SD). There was no significant difference in serum estradiol concentrations between cases (n = 136) and controls (n = 116). The CYP 19 genotype was significantly associated with serum estradiol (P = 0.002). Women with the AA genotype had higher serum estradiol concentrations compared with those with the GG genotype (P = 0.03). In older women, those with the CYP 19 GA and GG genotypes had an increased prevalence of osteoporosis (P = 0.04) and fractures (P = 0.003). We found no significant association between CYP 17 genotype and serum androgens and estradiol concentrations. However, a significant association was seen between BMD values at the femoral neck with CYP 17 genotype in cases (P = 0.04) and in the whole study population (P = 0.012). Subjects with the CC genotype had significantly lower BMD (mean +/- SD: TT, 0.7 +/- 0.16; CC, 0.6 +/- 0.08 g/cm(2); P = 0.006). In conclusion, both CYP 17 and CYP 19 are candidate genes for osteoporosis in postmenopausal women.

Apolipoprotein E genotype and Alzheimer's disease in Hong Kong elderly Chinese.

We studied the apolipoprotein E (apoE) allele frequencies in 65 Chinese patients with late-onset Alzheimer's disease (AD) and 82 age- and sex-matched controls. The apoE epsilon 4 allele frequency was significantly higher in the AD group than in the control group (0.169 versus, p < 0.01). There were five homozygotes for epsilon 4 in the AD group but none among the controls. The odds ratio for AD was 1.6 for epsilon 4 heterozygotes. The age at onset was lower in the presence of the epsilon 4 allele and higher with the epsilon 2 allele, although neither of these differences reached statistical significance. The association between apoE alleles and AD previously reported in Caucasian populations was also present in this reports of lower prevalence of AD compared with the prevalence of multi-infarct dementia.

Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin production by human osteoblastic cells: comparison of the effects of 17-beta oestradiol and raloxifene.

Oestrogen inhibits bone resorption, at least in part, by regulating the production of several cytokines, including interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) by cells of the osteoblastic lineage. The selective oestrogen receptor modulator raloxifene (RAL) acts on bone in a similar manner to oestrogen, although the mechanisms of action of RAL on osteoblasts still remain unclear. We investigated and compared the effects of 17-beta oestradiol (E(2)) and RAL on the regulation of IL-6, IL-1, RANKL and OPG in vitro in primary human osteoblastic (HOB) cells and in an immortalised clonal human bone marrow stromal cell line (HCC1) with osteoblastic characteristics. We tested E(2) and RAL at concentrations ranging from 10(-12) to 10(-6) M. IL-6, IL-1alpha and IL-1beta, OPG and RANKL were measured by ELISA. RANKL and OPG mRNA steady state level was assessed by quantitative PCR analysis. Both E(2) and RAL led to a significant reduction in IL-6 production in the HOB cells, although the effect was more marked with E(2) (P<0.05). IL-1alpha and IL-1beta also decreased significantly following treatment with E(2) and RAL in the HCC1 cells (E(2) (10(-8), 10(-7) and 10(-6) M), % reduction (means+/-S.E.M.) compared with vehicle-treated cells - IL-1alpha: 84+/-7.4, 70.8+/-2.9*, 78.2+/-4.8*; IL-1beta: 79+/-10, 72.8+/-8.2*, 66.6+/-2.8*; RAL (10(-8), 10(-7) and 10(-6) M) - IL-1alpha: 72.4+/-5*, 79+/- 5.2*, 102+/-7.7; IL-1beta: 67.9+/-3.2*, 69+/-2.5*, 73.8+/- 6.2*; *P<0.05). OPG protein concentration decreased significantly in a dose-dependent manner following treatment with E(2) and RAL (% reduction E(2) (10(-8), 10(-7) and 10(-6) M) - HOB: 72.5+/-8.4*, 80+/-6.7*, 62.8+/-8.9*; HCC1: 109+/-4, 98.8+/-6, 54.5+/-3.4*; RAL (10(-8), 10(-7) and 10(-6) M) - HOB: 81.5+/-5.5*, 62.7+/-7.4*, 55.2+/-10.9*; HCC1: 92.7+/-7.4, 67+/-12.2*, 39+/-4.5*; *P<0.05). In the HCC1 cells, RANKL protein did not change significantly following E(2). In contrast, a significant reduction in RANKL was seen with RAL at 10(-7) and 10(-6) M (66+/-6.4% and 74+/-3% respectively). There was no change in OPG mRNA expression following E(2) or RAL in the HCC1 cells, although in the HOB cells we observed a significant reduction in OPG mRNA. RANKL mRNA decreased significantly in the HCC1 cells following RAL (10(-8), 10(-7)and 10(-6) M) treatment (% change from controls: 52+/-2*, 62+/-1*, 53+/-5.8*; *P<0.05). Similar results were seen in the HOB cells with RAL at 10(-6) M (RANKL mRNA: 72+/-5.5, P<0.05). In addition, there was a significant decrease in the RANKL/OPG ratio after RAL at 10(-6) M (HOB: 65.6+/-5*, HCC1: 56.9+/-20*; *P<0.05). RANKL/OPG ratio did not change significantly in the HCC1 cells following E(2). However, in contrast to RAL, we observed an increase in the RANKL/OPG ratio in the HOB cells following treatment with E(2). In conclusion, the study shows that RAL and E(2) have divergent cell-specific effects on the regulation of cytokines. The data also suggest that, in contrast to E(2), RAL may exert its anti-resorptive actions, at least in part, via the RANKL/OPG pathway. Further in vivo studies are required to confirm this.

Biochemical investigation of young hospitalized Chinese children: results over a 7-year period.

During the seven years from January 1989 to December 1995, we investigated 2,269 Chinese infants and young children for metabolic disorders in Hong Kong. These young patients, all aged under 4 years and originated from southern China, were ill with no apparent cause and had clinical manifestations suggestive of inherited metabolic diseases. A spot urine and a plasma sample were obtained from each patient for biochemical analysis, including urinary organic acid identification and plasma amino acid analysis. Six cases of mucopolysaccharidosis, four multiple carboxylase deficiency, three 2-methylacetoacetyl CoA thiolase deficiency, two methymalonic aciduria, one glutaric aciduria type I, one glutaric aciduria type II, one a-oxoglutaric aciduria, and one case of orotic aciduria were detected. There were also single suspected cases of medium-chain acyl-CoA dehydrogenase deficiency and isovaleric aciduria. No primary amino acid disorder, such as phenylketouria and maple syrup urine disease, has been detected. Our results suggest that a different pattern of inherited metabolic diseases exists in the southern Chinese when compared with the Chinese in other regions of China.

Biochemical predictors of short term mortality in elderly residents of chronic care institutions.

A survey of 208 elderly subjects living in four long term care institutions was undertaken over three months to identify nutritional and other variables that could be used to predict mortality during the subsequent three months. There were 58 men (mean age (SD) 75.6 (9.6) years) and 150 women (79.5 (8.4) years). Twenty nine subjects died (12 men and 17 women) within three months of completing the study. Twenty eight out of 57 variables differed significantly between those who died and those who survived. Subjects who died had lower systolic blood pressure, poorer intake of protein calories, lower concentrations of haemoglobin, plasma retinol, zinc, total cholesterol, and higher albumin adjusted plasma calcium concentrations. Stepwise regression analysis identified five variables that predicted mortality: plasma fructosamine; transferrin; glycosylated haemoglobin; prealbumin; and haemoglobin. The sensitivity, specificity, and predictive values of the discriminant function score using 0 as the demarcation between survivors and non-survivors were 75%, 97%, and 95%, respectively. This score could therefore be used to identify those most in need of nutritional support.

Gas chromatographic-mass fragmentographic determination of serum 1 alpha,25 dihydroxyvitamin D3.

We extracted 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] from 10 mL serum using Sep-Pak C18 and Sep-Pak Silica mini-columns and normal-phase high performance liquid chromatography (HPLC) separation for analysis by gas chromatography-mass fragmentography (GC-MS). A GC-MS method was optimised using manual tuning for ion mass calibration and selective ion monitoring (SIM) for quantitation. Serum 1 alpha,25(OH)2D3 was identified by superimposition of the m/z 452 and 501 ion peaks and by overlapping the m/z 452 ion peak with that of its authentic standard. It was quantitated from the relative peak areas of its m/z 452 ion and the m/z 363 ion of vitamin D2, the internal standard. Twenty picograms of 1 alpha,25(OH)2D3 gave a peak with a signal-to-noise ratio of 26:1. Between-batch coefficient of variation (CV) for 1 alpha,25(OH)2D3 standard was < 13%. However, serum analysis was less precise, within-batch CV being 20%. The analytical recovery was about 70% and detection limit 0.5 pg/mL. When compared with a commercial radioreceptor assay we still found our method to be sensitive, specific, and adequate for confirmative and semiquantitative analysis of serum 1 alpha,25(OH)2D3.

No association detected between very-low-density lipoprotein receptor (VLDL-R) and late-onset Alzheimer's disease in Hong Kong Chinese.

The epsilon4 allele of apolipoprotein E (ApoE) is a risk factor in late-onset Alzheimer's disease (AD). As a receptor for ApoE, very-low-density lipoprotein receptor (VLDLR) might be involved in AD pathogenesis. A Japanese study [Okuizimi, K., et al., Nature Genet., 11 (1995) 207-209] has shown an increased 5 and decreased 8 CGG-repeat allele frequency in the 5' untranslated region of VLDLR in Japanese AD versus normal controls (N). Subsequent studies in Caucasian Americans failed to duplicate the result. We examined this polymorphism in pathologically- or clinically-diagnosed Chinese late-onset AD. Our data did not show a significant increase in the 5 CGG-repeat in AD, thus suggesting no association to VLDLR. However, our data did show that the allele frequencies for each CGG-repeat were similar in both Chinese and Japanese.

Low density lipoprotein receptor related protein gene exon 3 polymorphism association with Alzheimer's disease in Chinese.

Since apolipoprotein E4 (apoE4) is the major genetic risk for late onset Alzheimer's disease (AD), proteins that interact with apoE might be involved in AD pathogenesis. Low density lipoprotein receptor related protein (LRP) is an apoE receptor in the brain. In exon 3 of the LRP gene a polymorphism was found to be underrepresented in AD compared to normal Caucasian subjects (N). We examined this polymorphism in Chinese AD and N subjects. The polymorphism frequency in N was roughly half that reported for Caucasians. Compared to N, the frequency was significantly decreased in pathologically diagnosed, but not in clinically diagnosed AD patients. Thus, the role of the LRP exon 3 polymorphism in AD has now been demonstrated in two ethnic groups, suggesting the importance of LRP in AD pathogenesis.

Intake and blood levels of some minerals in an elderly Chinese population.

Twenty-four hour intakes of potassium, calcium, and iron, and plasma concentration of potassium, copper and zinc were determined in a cluster sample of 400 apparently healthy subjects aged 60 years and over living in the community. Potassium and calcium intakes were lower in older men and lower in men compared to women. Both were much lower than intakes reported among Caucasian elderly, while iron intakes are comparable. Plasma potassium concentration was lower than values from studies of all ages and from studies of Caucasian elderly subjects. Copper and zinc concentrations were comparable to published values for adults of all ages. The significance of low potassium and calcium intake among elderly subjects in the population is discussed.

Vitamin A and E status in healthy elderly Chinese in Hong Kong.

Daily intakes of retinol and beta-carotene equivalents, plasma retinol, retinol binding protein and plasma vitamin E were estimated in a cluster sample of apparently healthy elderly subjects aged 60 years and over in Hong Kong leading an active life in the community. Intakes were available from 418 and plasma measurements from 385 subjects. There were no age or sex differences in intakes. Plasma retinol and retinol binding protein levels showed no sex- or age-related difference, while plasma vitamin E levels were higher in women. Retinol binding protein, plasma retinol and plasma vitamin E levels were comparable to published values for Chinese and Caucasians of all ages. Vitamin A and E status appears to be adequate in this elderly Chinese population.

Protein nutritional status in elderly Chinese in Hong Kong.

Daily intakes of protein and energy were estimated in a group of 417 apparently healthy elderly Chinese subjects aged 60 years and over, living independently in the community. The mean protein intake was 1.2 g/kg body weight, well above the WHO/FAO/UNU recommendation of 0.8 g/kg, and comparable to intakes of elderly Americans and Britons. Plasma total protein, albumin, prealbumin, retinol binding protein and transferrin concentrations were also measured. No age- or sex-related differences were found and the values were comparable to published values for elderly Caucasians. Plasma prealbumin and retinol binding protein concentrations correlated with protein intake and with arm muscle area. Protein nutritional status appears adequate for elderly Chinese living in the community.

Nutritional status of the water-soluble vitamins in an active Chinese elderly population in Hong Kong.

Dietary intakes of thiamine, riboflavin, nicotinic and ascorbic acid, together with the biochemical status of thiamine, riboflavin, pyridoxine and ascorbic acid, were determined in a cluster sample of 419 healthy active elderly subjects aged 60 years and above living in the community. Nicotinic acid intake per 1000 kcal (4.18 MJ) of food energy showed an age-related decrease in men, while women had higher ascorbic acid intakes than men. Between 38 and 98 per cent of this population have intakes of thiamine, riboflavin and nicotinic acid below the UK RDA values. Intakes of ascorbic acid were below the RDA for 17 per cent of men and 9 per cent of women. The prevalence of biochemical deficiency was 8, 14, 11.5 and 24 per cent for thiamine, riboflavin, pyridoxine and ascorbic acid respectively. A significant difference in intakes between groups with blood levels within and below the reference range was seen only for riboflavin, suggesting that factors other than low intake may be more important in contributing to low blood levels for thiamine and ascorbic acid. However, inaccuracies in dietary intake estimations may contribute to the poor correlation.

Automated trichloroacetic acid precipitation method for urine total protein.

Urine total protein determination by a trichloroacetic acid (TCA) precipitation method was automated on a Cobas Bio centrifugal analyser. The assay measures the turbidity at 420 nm when a 50 microL sample is mixed with 150 microL of 30 g/L TCA at 25 degrees C. Samples up to 5 g/L can be measured by this method and interference due to pigments and turbidity can be minimised by running a blank with 1.25% hydrochloric acid (HC1) instead of TCA. The standard curve is stable and can be stored in the microcomputer and used again. Within-assay precision (CV) varied from 2 to 4.6% and between-assay precision varied from 1.8 to 5.4%. Analytical recovery ranged from 95 to 106.5% and the results correlated well with those obtained by a manual TCA method (r = 0.98). The method is easy to perform and is considerably faster than the manual procedure, thus saving time and providing a faster turnaround time.

Plasma and erythrocyte zinc concentrations in pre-eclampsia.

Plasma and erythrocyte zinc concentrations were measured in 28 Chinese pre-eclamptic women and 28 controls matched for parity, race and gestation. There were no differences in either the plasma or erythrocyte zinc concentrations between pre-eclamptic and control groups, although the mean birth weight (p less than 0.001) and period of gestation (p less than 0.001) at delivery in the control group were significantly higher. In the pre-eclamptic patients, those delivering before 37 weeks or those who gave birth to low birth weight (less than 2500 g), babies had a significantly higher plasma urate concentration (p less than 0.02) compared to the pre-eclamptic patients with better fetal outcome. However, the plasma and erythrocyte zinc concentrations between these subgroups were not significantly different. Our results suggest that zinc deficiency is unlikely to play a significant role in pre-eclampsia in our patients, and that measurement of plasma and erythrocyte zinc concentrations is of doubtful clinical value in the management of pre-eclampsia.

Permanent deficits in brain functions caused by long-term ketamine treatment in mice.

Ketamine, an injectable anesthetic, is also a popular recreational drug used by young adults worldwide. Ketamine is a non-competitive antagonist of N-methyl-d-aspartate receptor, which plays important roles in synaptic plasticity and neuronal learning. Most previous studies have examined the immediate and short-term effects of ketamine, which include learning and cognitive deficits plus impairment of working memory, whereas little is known about the long-term effects of repeated ketamine injections of common or usual recreational doses. Therefore, we aimed to evaluate the deficits in brain functions with behavioral tests, including wire hang, hot plate and water maze tests, plus examine prefrontal cortex apoptotic markers, including Bax, Bcl-2 and caspase-3, in mice treated with 6 months of daily ketamine administration. In our study, following 6 months of ketamine injection, mice showed significant deterioration in neuromuscular strength and nociception 4 hours post-dose, but learning and working memory were not affected nor was there significant apoptosis in the prefrontal cortex. Our research revealed the important clinical finding that long-term ketamine abuse with usual recreational doses can detrimentally affect neuromuscular strength and nociception as part of measurable, stable and persistent deficits in brain function.

How would composite traditional Chinese medicine protect the brain--an example of the composite formula "Pien Tze Huang".

Chinese medicine has a long history of several thousand years. The main form of Traditional Chinese Medicine (TCM) is composite, i.e. a mixture of up to 10 medicinal products. Thus a composite prescription of 4-5 kinds of Chinese medicinal products may contain several hundred kinds of chemical composition. The active ingredients and clinical efficacy of which are difficult to characterize. We aim to review the Chinese literature of TCMs with neuroprotective effects. We illustrate with our study on Pien Tze Huang (PZH) the use of in vivo tests in the study of composite TCM. Our results show evidence that PZH might have neuropreventive effects in rats.

Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine.

Ketamine, a non-competitive antagonist at the glutamatergic N-methyl-d-aspartate (NMDA) receptor, might impair memory function of the brain. Loss of memory is also a characteristic of aging and Alzheimer's disease. Hyperphosphorylation of tau is an early event in the aging process and Alzheimer's disease. Therefore, we aimed to find out whether long-term ketmaine administration is related to hyperphosphorylation of tau or not in the brains of mice and monkeys. Results showed that after 6 months' administration of ketamine, in the prefrontal and entorhinal cortical sections of mouse and monkey brains, there were significant increases of positive sites for the hyperphosphorylated tau protein as compared to the control animals receiving no ketamine administration. Furthermore, about 15% of hyperphosphorylated tau positive cells were also positively labeled by terminal dUTP nick end labeling (TUNEL) indicating there might be a relationship between hyperphosphorylation of tau and apoptosis. Therefore, the long-term ketamine toxicity might involve neurodegenerative process similar to that of aging and/or Alzheimer's disease.