Pokkuri Death Syndrome; sudden cardiac death cases without coronary atherosclerosis in South Asian young males.

Sudden death is one of the major concerns in forensic medicine. Especially when the deceased is a young subject without significant history, the case will be of major interest to the authorities. Sudden unexplained cardiac death has been known as "Pokkuri Death Syndrome" (PDS) in Japan, "Lai Tai" in Thailand, "Bangungut" in the Philippines, "Dream Disease" in Hawaii, and "Sudden Unexpected Nocturnal Death Syndrome" among South Asian immigrants in the USA. However, the clinical and pathological features of these sudden death cases, especially the characteristics of no coronary atherosclerosis, are surprisingly similar and mainly occur among Southeast Asian young males during sleep in the midnight. In this manuscript, we have reviewed the pathological characteristics and the possible mechanism of death in PDS cases, which were associated with significantly elevated remnant lipoproteins in plasma as revealed from our studies during the past 15 years in Japan. Although elevated plasma remnant lipoproteins have been known to be strongly atherogenic, coronary atherosclerosis was not observed in PDS cases. PDS cases were shown to be an interesting cardiovascular disease death discovered in forensic medicine research, which may suggest the difference between the occurrence of cardiovascular events and the severity of coronary atherosclerosis as separate factors. These observations in PDS cases suggest the possibility that the intervention could be more targeted to suppress the cardiovascular events rather than to slow down the progression of atherosclerosis, which is now most extensively targeted for the therapy of cardiovascular disease in Western countries.

Detection of apolipoproteins B-48 and B-100 carrying particles in lipoprotein fractions extracted from human aortic atherosclerotic plaques in sudden cardiac death cases.

BACKGROUND: ApoB-48 is a major apolipoprotein secreted by the small intestine and is the main constitutive apolipoprotein in chylomicrons (CM). In the past, presence of apoB-48 in human aortic atherosclerotic plaques has not been detected. METHODS: A newly developed apoB-48 ELISA together with an HPLC fractionation technique, were applied to investigate the presence of apoB-48 (CM) in aortic atherosclerotic plaques. The atherosclerotic plaques were obtained from aortae of sudden cardiac death cases. Total cholesterol, triglycerides (TG), apoB-100 and apoB-48 were measured in the aortic plaques extracts. RESULTS: HPLC analysis of plaques extracts monitored by cholesterol revealed mainly particle sizes of CM and very low density lipoproteins (VLDL) in the d>1.006 fractions. The plaques extracts were monitored by apoB-48 and apoB-100 ELISA. There were no TG peaks in any lipoprotein fraction extracted from the plaques except as free glycerol. ApoB-100 was detected in VLDL particles and in LDL sizes. In contrast, apoB-48 was detected in particles of CM, VLDL and LDL sizes. Further, in postmortem plasma, apo B-48 was detected in particles sizes of HDL or smaller and the Western blot analysis could not show any 250 kDa molecular weight (MW) protein in the plaque extracts, but smaller and broader MW staining were observed at 20-150 kDa. CONCLUSION: Hitherto there has been lack of an appropriate assay to measure apoB-48 in plaques. Our investigations show that apoB-48 is present in atherosclerotic plaques with denatured or degraded structure. This is the first report describing presence of apoB-48 in human atherosclerotic plaques.

Plasma remnant-like lipoprotein particles or LDL-C as major pathologic factors in sudden cardiac death cases.

We have previously reported that the majority of sudden cardiac death (SCD) events were associated with postprandial hyperlipidemia in Japanese subjects. In this investigation, we have compared LDL-cholesterol (LDL-C) and remnant-like lipoprotein particles (RLP) as cardiovascular risk factors in SCD cases, especially in Pokkuri death syndrome (PDS) cases who had nearly normal coronary arteries. To predict the risk of plasma RLP-cholesterol, triglyceride (RLP-C, RLP-TG) and LDL-C in fatal clinical events associated with SCD cases with or without atherosclerosis (PDS), we calculated the cut-off values and likelihood ratio of these lipoproteins from ROC analysis. Sixty-eight percent of SCD cases were above cut-off value of RLP-C (>12.8 mg/dL) versus 32% for control death cases (P<0.0001) and the likelihood ratio of RLP-C was 2.12. Significantly higher incidence of RLP-C above cut-off value (>10.1 mg/dL) was seen in PDS compared to controls (P<0.0001) and the likelihood ratio was 3.13. Similarly, significantly higher incidence of RLP-TG above cut-off values, SCD>53 mg/dL and PDS>67 mg/dL, was seen compared to controls (P<0.0001) and the likelihood ratio was 1.86 and 2.73, respectively. Further, significantly higher incidence of LDL-C above cut-off value (>93 mg/dL) was seen in SCD compared to controls (P<0.0001) and the likelihood ratio was 1.68. However, the incidence of LDL-C above cut-off value (LDL-C>106 mg/dL) was not significantly different between PDS and controls and the likelihood ratio was 1.52. In conclusion, this study has shown high levels of plasma remnant lipoproteins in PDS and that PDS cases did not present with atherosclerotic lesions or elevated LDL-C. In contrast, SCD cases showed high levels of plasma remnant lipoproteins together with elevated plasma LDL-C. Accordingly, we believe that plasma remnant lipoproteins level rather than plasma LDL-C is a major pathologic factor in cardiovascular events.

Rapid simultaneous determination of ephedrines, amphetamines, cocaine, cocaine metabolites, and opiates in human urine by GC-MS.

This paper presents a simple and sensitive chromatographic procedure for the simultaneous determination and quantification of ephedrines, amphetamines, cocaine, cocaine metabolites, and opiates in human urine by gas chromatography-mass spectrometry. This method involves enzyme hydrolysis in the presence of a deuterated internal standard, liquid-liquid extraction, and derivatization with pentafluoropropionic anhydride and pentafluoropropanol. The recovery of each compound averaged at 65.8% or more. The limits of detection determined for each compound by using a 2-mL sample volume ranged from 5 to 50 ng/mL. The calibration curves were linear to 100 ng/mL for all compounds when determined using methamphetamine-d4 and MDMA-d5 as internal standards. This method was successfully applied for the analysis of urine samples suspected to contain intoxicants such as methamphetamine and heroin.

ApoB-100 carrying lipoprotein, but not apoB-48, is the major subset of proatherogenic remnant-like lipoprotein particles detected in plasma of sudden cardiac death cases.

We have previously reported that plasma levels of remnant-like lipoprotein particles (RLP) significantly increased in sudden cardiac death cases with and without coronary atherosclerosis. In this study we have elucidated the major subset of proatherogenic RLP, containing both apoB-48 and apoB-100-carrying remnants, in plasma of SCD and control death cases. One hundred and sixty seven Japanese cases of sudden cardiac death and 78 cases of control death underwent autopsy within 12h after death were studied. Heart weight was 9.2% higher in SCD cases than controls (P<0.05). Moreover 57.5% or 96/167 of the cases had more than grade (2+) coronary atherosclerosis versus 21.8% or 17 of 78 controls (P<0.01). Approximately 2/3 of the cases had full stomach, reflecting the postprandial state at the time of death. Plasma TC, TG, VLDL-C, LDL-C were significantly elevated (P<0.001) together with RLP-C (P<0.01), RLP-TG (P<0.005) in SCD cases. Plasma RLP-apoB-100 levels were significantly elevated in SCD (P<-0.001), but apoB-48 levels were not. The median ratio of apoB-100/apoB-48 in RLP was 7.1 in SCD. The median RLP-TG/RLP-C ratio was 4.7, which suggested a large VLDL size. When apoB-48 and apoB-100 in RLP were divided into two groups, above and below the median level, respectively, apoB-48 inversely correlated with RLP-C (P<0.05) and RLP-TG (P<0.01), while apoB-100 in RLP positively correlated with RLP-C (P<0.01) in SCD cases. In conclusion, these results indicated that apoB-100 carrying lipoproteins, not apoB-48 carrying lipoproteins, were the major subset of RLP associated with sudden cardiac death in the postprandial state, regardless to the severity of coronary atherosclerosis.

Sudden death in a patient with idiopathic scoliosis.

We report an autopsy case of sudden death in a 36-year-old craftsman with idiopathic scoliosis. The doctor identified his scoliosis at the age of thirteen, and he was under medical care for three years until he stopped consulting the doctor. He collapsed while walking at the station and was sent to an emergency room in cardiopulmonary arrest state, where he was declared dead in spite of more than an hour of CPR. Numbers of petechiae were seen on the bilateral palpebral conjunctivae and the lips were cyanotic. There were no particular injuries except for small abrasions observed on the face. The back showed right rib hump owing to midthoracic scoliosis (with 73 degrees of Cobb's angle) and right hemithorax was deformed showing an appearance of pectus excavatum in the front. The volume of the right thoracic cavity was significantly decreased. In the right lung, there was extensive stromal fibrosis, leaving almost no normal alveolar structures, and medial hypertrophy of pulmonary arteriolar walls. Hypertrophy of the right heart ventricle due to these pulmonary changes and the congestion of other organs suggested that the cause of death in this case was cor pulmonale due to pulmonary hypertension. This was a rare case of fatal outcome of advanced idiopathic scoliosis without medical care in spite of early detection through mass screening.

The lipid and lipoprotein profiles of apo B-carrying particles in remnant-like lipoproteins isolated from plasma in sudden cardiac death cases.

OBJECTIVE: Remnant-like lipoprotein particles (RLP) are known as a subset of TG-rich lipoproteins which possess proatherogenic properties. In this study we have elucidated the characteristics of apoB carrying lipoprotein particles in RLP isolated from plasma in sudden cardiac death (SCD) cases and in coronary artery disease (CAD) patients. METHODS: Lipids and lipoproteins, especially apoB-48 and apoB-100, were analyzed in plasma and in RLP from SCD and CAD cases with full stomach. RESULTS: SDS-PAGE and ELISA showed the predominance of apoB-100 in RLP compared with apoB-48. The main peak of RLP fractionated by HPLC and monitored by TC and TG revealed the particle size at VLDL or IDL, similar to those from CAD patients. The main peak of RLP monitored by apoB-48 revealed the particle size smaller than those monitored by apoB-100 in both cases. RLP-TG/total TG ratio was shown to be significantly higher in SCD cases than in control death cases (P < 0.001) in spite of the same fatty acid composition in RLP between the cases. CONCLUSION: These results suggested that the major subset of apoB particles in RLP isolated from plasma in SCD cases was apoB-100 carrying VLDL remnants, not apoB-48 CM remnants, which was identical with RLP in CAD patients.

Validated gas chromatographic-negative ion chemical ionization mass spectrometric method for delta(9)-tetrahydrocannabinol in sweat patches.

BACKGROUND: A sensitive gas chromatography-negative ion chemical ionization mass spectrometry (GC/MS-NICI) method was developed and validated for the measurement of Delta(9)-tetrahydrocannabinol (THC) in human sweat patches. METHODS: THC-d(0) and THC-d(3) were added to worn blank sweat patches (PharmChek; PharmChem Incorporated) and extracted with 3 mL of methanol-0.2 mol/L sodium acetate buffer (pH 5.0, 3:1 by volume) on a reciprocating shaker at ambient temperature for 30 min. Extracted solution (2 mL) was diluted with 8 mL of 0.1 mol/L sodium acetate buffer (pH 4.5) and extracted by use of solid-phase extraction columns (CleanScreen; United Chemical Technologies). Dried extracts were derivatized with trifluoroacetic acid and analyzed with an Agilent 6890 gas chromatograph interfaced with an Agilent 5973 mass selective detector operated in NICI-selected ion-monitoring mode. RESULTS: The lower limits of detection and quantification for THC in human sweat were 0.2 and 0.4 ng/patch, respectively. The calibration curve was linear from 0.4 to 10 ng/patch (R(2) >0.995). Overall recovery of THC from blank worn patches to which 0.6, 4.0, and 8.0 ng of THC had been added was 44-46%. Assay imprecision, expressed as CV, was <10% for 0.6, 4.0, and 8.0 ng/patch quality-control samples. Twenty-one potential interfering compounds (50 ng/patch) added to low quality-control samples (0.6 ng/patch) did not influence THC quantification. CONCLUSIONS: This GC/MS-NICI assay for THC in human sweat provides adequate sensitivity and performance characteristics for analyzing THC in sweat patches and meets the requirements of the proposed Substance Abuse and Mental Health Administration's guidelines for sweat testing.

Validity of plasma remnant lipoproteins as surrogate markers of antemortem level in cases of sudden coronary death.

Hitherto triglycerides (TG) and TG-rich lipoproteins were been of limited value as surrogates for antemortem levels. We measured TG levels in postmortem plasma from sudden coronary death cases (SCD, n=91) by using two TG assays, Dry Chem TG (free glycerol was added) and the Determiner L-TG (without added free glycerol) that measured net TG. TG levels were markedly higher by the Dry Chem TG (y) vs. Determiner L-TG (x), y = 1.03x + 229 mg/dl. HPLC showed large amounts of free glycerol in postmortem plasma and in TG-rich lipoprotein remnants (RLP). These results were verified in a rabbit model of SCD. Further, RLP from SCD were found to be biophysically similar to those from living patients with coronary artery disease (CAD). In conclusion, postmortem plasma sampled up to 12 h after death is appropriate for measuring lipid and lipoproteins, TG and RLP-TG as surrogates for antemortem levels when a TG assay without added free glycerol is used.

Remnant lipoproteins from patients with sudden cardiac death enhance coronary vasospastic activity through upregulation of Rho-kinase.

OBJECTIVE: Sudden cardiac death (SCD) still remains a serious problem. We have previously shown that remnant-like particles (RLP) are the major risk factor for SCD and that Rho-kinase plays a central role in the molecular mechanism of coronary vasospasm. In this study, we examined whether RLP from patients with SCD upregulate Rho-kinase associated with an enhanced coronary vasospastic activity. METHODS AND RESULTS: We isolated RLP and non-RLP in very-low-density lipoprotein (VLDL) fraction from SCD patients without coronary stenosis. We performed in vivo study in which we treated the coronary artery with RLP or non-RLP fraction at the adventitia in pigs. After 1 week, intracoronary serotonin caused marked coronary hyperconstriction at the segment treated with RLP fraction but not with non-RLP fraction (P<0.001, n=6), and hydroxyfasudil, a selective Rho-kinase inhibitor, dose-dependently inhibited the spasm in vivo. In organ chamber experiments, serotonin caused hypercontraction of vascular smooth muscle cells (VSMC) from RLP-treated segment, which was significantly inhibited by hydroxyfasudil (P<0.001, n=6). In cultured human coronary VSMC, the treatment with RLP significantly enhanced the expression and activity of Rho-kinase (P<0.05, n=6). CONCLUSIONS: These results indicate that RLP from SCD patients upregulate Rho-kinase in coronary VSMC and markedly enhance coronary vasospastic activity.

Hypertriglyceridemia in pregnancy does not contribute to the enhanced formation of remnant lipoprotein particles.

BACKGROUND: During pregnancy, serum cholesterol (TC) and triglyceride (TG) concentrations are known to increase significantly, but whether remnant lipoprotein particles (RLP) increase has not been shown. METHODS: We compared lipid profiles in 22 healthy pregnant women to 31 healthy nonpregnant women and 24 patients with diabetes mellitus (DM), by measuring cholesterol and TG concentrations in major lipoprotein classes after HPLC separation and immunoseparation of RLP. RESULTS: Serum TG and TC concentrations were significantly higher in the pregnant group than in the healthy control or DM groups. Cholesterol and TG concentrations of all major lipoprotein classes were also significantly higher in the pregnant group than the control and DM groups, except for VLDL-TG in the DM group. RLP-C and RLP-TG concentrations were significantly higher in the pregnant group (8.7 mg/dl and 25.4 mg/dl on average) than the control group (2.4 mg/dl and 5.7 mg/dl), but not different from the DM group (8.8 mg/dl and 24.1 mg/dl). RLP-TG to RLP-C ratios were similar among the three groups and correlated with the VLDL-TG to VLDL-C ratio. The percentages of RLP-C in VLDL-C and RLP-TG in VLDL-TG in the pregnant group (15.9% and 15.7%) were significantly lower than those of the control (48.5% and 35.6%) and the DM (32.7% and 20.8%) groups. CONCLUSIONS: RLP increased moderately during gestation with the increase in VLDL and TG, but the percentage of RLP in VLDL was significantly lower in the pregnant women compared with the control and DM patients, suggesting that hypertriglyceridemia in pregnancy is not primarily due to an increase in the atherogenic RLP.

Remnant-like particles from subjects who died of coronary artery disease suppress NO synthase activity and attenuate endothelium-dependent vasorelaxation.

BACKGROUND: Plasma levels of remnant-like particles (RLP) is one of the predictive markers for coronary artery disease (CAD), and the inhibition by RLP of endothelium-dependent vasorelaxation has been reported. We attempted to clarify whether or not RLP, which inhibits endothelium-dependent vasorelaxation, affects nitric oxide (NO) production and NO synthase (eNOS) levels in cultured endothelial cells. METHODS: RLP were obtained from postmortem blood of subjects who died of CAD. Modification by RLP of acetylcholine-induced relaxation of rabbit aorta, and changes in NO production and (eNOS) in cultured bovine endothelial cells were examined. RESULTS: RLP at 750 and 1500 microg triglyceride/ml inhibited vasorelaxation, and at 5-160 microg triglyceride/ml, concentration-dependently inhibited NO production. However, (eNOS) did not decrease after incubation with RLP. CONCLUSION: Postmortem RLP from subjects who died of CAD do not change the amount of (eNOS), but rather, inhibits its activity and attenuates endothelium-dependent vasorelaxation.

Haploid allele mapping of Y-chromosome minisatellite, MSY1 (DYF155S1), to a Japanese population.

The present study analyses the human Y-chromosome minisatellite locus, MSY1 (DYF155S1), in 205 Japanese males of 191 pedigrees using the minisatellite variant repeat (MVR) mapping system. The internal haploid structures of the detected alleles considerably varied and consisted of three major repeat units: types 2, 3 and 4. A comparison of the haploid profiles of the MVR codes identified 185 distinct alleles, of which only five were shared. We did not detect a type 1 repeat unit, and variations were frequent at the 5' end of the minisatellite locus. Within an analysis of 24 paternally linked DNA samples donated by ten families, no mutational events were identified even over two generation gaps. Furthermore, we applied this mapping system to a paternity test in which the alleged father was missing.

Fatal subarachnoid hemorrhage complicating actinomycotic meningitis.

Actinomycosis is caused by Gram-positive Actinomyces species that are part of the normal oral flora with low virulence. We describe a rare case of sudden death of a 48-year-old man with actinomycotic basilar meningitis that was complicated by fatal subarachnoid hemorrhage. Autopsy revealed meningitis at the basilar region of the brain, and histological examination revealed characteristic bacterial aggregates with extensive leukocyte infiltration and severe vasculitis of arteries of this region. Rupture of an artery by severe arteritis was thought to be the cause of the subarachnoid hemorrhage. The probable primary source of infection was found in the left lung. To the best of our knowledge, the complication of subarachnoid hemorrhage has not been reported previously in actinomycotic meningitis.

Hippuric acid and methyl hippuric acid in rat hair: possible monitoring of xylene and toluene exposure.

Thinner is mainly composed of toluene and xylenes, and we studied the incorporation of the main metabolites of toluene and xylenes, hippuric acid (HA) and o-, m-, and p-methyl hippuric acids (o-, m-, p-MHA), in dark agouti rats' hair. Rat black hair was shaved before any exposure with an electric shaver designed for animals. Studies were performed in vivo with exposures of 30 min per day at three different concentrations (100, 300, and 1000 ppm) of toluene and o-, m-, and p-xylene for a total of 10 times over 2 weeks. Newly grown hair was tweezed out from the root with tweezers at seventh of the last exposure. Hair samples were then washed, extracted, derivatized, and analyzed by gas chromatography-mass spectrometry (GC-MS). HA and o-, m-, and p-MHA were not detected (ND) in the unexposed rat hair. After exposure, the metabolite concentration in the hair changed depending on the exposure concentration. Mean concentrations ranged from ND to 7.6 ng/mg, from ND to 13.8 ng/mg, from ND to 10.1 ng/mg, and from ND to 9.2 ng/ml hair for HA, o-, m-, and p-MHA, respectively. These results indicate that the metabolites concentrations in hair are effective indices of thinner exposure.

Headspace solid-phase micro-extraction and gas chromatography with micro-electron capture detection for the measurement of p,p'-DDE in rat whole blood and hair.

A sensitive, specific, and reproducible capillary gas chromatography (GC) with micro-electron capture detection (micro-ECD) method using headspace solid-phase micro-extraction (HS-SPME) for the quantitative analysis of p,p'-DDE in rat whole blood and hair was developed. A 100 microm polydimethylsiloxane (PDMS) phase was used for the extraction. The obtained detection limits were 0.003 ng/mg and 0.004 ng/mg in whole blood and hair, respectively, at a signal-to-noise ratio of 3 (S/N = 3). Linearity was obtained in the ranges 0.003-2.0 ng/mg and 0.004-2.0 ng/mg in whole blood and hair, respectively, and the correlation coefficients of whole blood and hair were greater than 0.998. This method was successfully applied to the analysis of p,p'-DDE in rat whole blood and rat hair after an oral dose of p,p'-DDE.

Temporal changes in testis weight during the past 50 years in Japan.

The ever-increasing presence of environmental toxicants and their disruptive effect on the reproductive systems of wildlife raises the question of possible damage to the human reproductive system. Using medicolegal data from over 20,000 Japanese men subjected to necropsy from 1948 to 1998, we investigated temporal changes in testis weight to find possible evidence of male reproductive disorders. We also carried out a histological examination of 747 testes collected from 1978 to 1998. Our detailed analyses of the development of testis weight over the past 50 years have revealed four clear phenomena: (i) the age at which testis weight reaches its maximum has decreased; (ii) peak weight showed a general increase until it started to decline in boys born after 1960; (iii) the decline-rate at which testis weight decreases after its peak has greatly accelerated; and (iv) the onset of increasing testis weight of boys has occurred at a progressively younger age. Our quantitative analyses of testis weight indicate the possibility of a subtle reproductive disorder in Japanese men, especially in those born after 1960. Together with the accelerated development and decline in testis weight during the past 50 years, the decline in peak weight might be indicative of a subtle interference of environmental toxicants with male reproductive organogenesis.

Simultaneous detection of hippuric acid and methylhippuric acid in urine by Empore disk and gas chromatography-mass spectrometry.

A method is described for the determination of hippuric acid (HA) and o-, m-, and p-methylhippuric acids (o-, m-, p-MHAs) in urine using solid-phase extraction and gas chromatography-mass spectrometry (GC-MS). The extraction procedure uses an Empore disk, derivatized into the respective trimethyl silyl derivatives. All metabolites including the internal standard (I.S.) were clearly able to be analyzed by the DB-17 column. The calibration curves for the four acids show linearity in the range of 5-70 microg/ml. The detection limit of each acid was 1.0-2.5 microg/ml.