BLOOD / SERUM MAGNESIUM, ZINC, COPPER AND
SELENIUM CONCENTRATIONS IN PATIENTS WITH
MYOCARDIAL INFARCTION OR RECEFVING DIGOXIN
Thesis submitted for
MSc in Clinical Biochemistry
Department of Chemical Pathology
The Chinese University of Hong Kong
By
XL\0 GANG
March, 1998 UL
j ^^^^|L_M^|
Ng^ UNIVERSITY J_ ^@>^BRARY SYSTEMZ^ x^^^ CONTENTS
ACKNOWLEDGEMENTS i
SUMMARY 1
1 LITERATURE REV正W
1.1 MAGNESKJM 3
1.1.1 GENERAL FUNCTION OF MAGNESIUM 3
1.1.2 ABSORPTION AND METABOLISM OF
MAGNESIUM 6
1.1.3 CLlMCAL ASPECTS 8
1.1.4 METHODS OF DETERVUNATlON OF
MAGNESRJM 11
1.2ZD^C 13
1.2.1 GENERAL FUNCTION OF ZWC 13
1.2.2 ABSORPTION AND METABOLISM OF ZESlC 14 ‘
1.2.3 CLEMICAL ASPECTS 16
1.2.4 ASSESSMENT OF THE BODY ZINC STATUS 19
1.2.5 METHODS OF DETERNGNATION OF ZENfC 20
1.3 COPPER 21
1.3.1 GENERAL FUNCTION OF COPPER 21
iii 1.3.2 ABSORPTION AND METABOLISM OF COPPER 21
1.3.3 OJNICAL ASPECT 25
1.3.4 LABORATORY ASSESSMENT OF
COPPER STATUS 28
1.3.5 METHODS FOR THE DETERJvONATION OF
COPPER 29
1.4 SELENRJM 31
1.4.1 GENERAL FUNCTION OF SELENIUM 31
1.4.2 ABSORPTION AND METABOLISM OF
SELENRJM 31
1.4.3 CLmiCAL ASPECTS 34
1.4.4ASSESSMENT 36
1.4.5 METHODS OF DETERMBMATION OF
SELENRJM 37
1.5 nsTTRODUCTION TO ATOMIC ABSORPTION
SPECTROPHOTOMETRY 38
1.5.1 PFONCIPLE OF AAS 38
1.5.2 mSTRUMENTATION 39
2 mnODUCTION 43
iv 2.1 TRACE ELEMENT DEFICffiNCY DM
HOSPITAL PATIENT 43
2.2 MICRONUTRIENT DEFICIENCY AMONG PATIENTS
WITH ACUTE MYOCARDIAL •ARCTION 44
2.2.1 MAGNESIUM DEFICIENCY 44
2.2.2 CHANGE OF ZINC AND COPPER IN AMI 47
2.2.3 SELENIUM DEFICIENCY 50
2.3 OBJECTIVES OF THIS PROJECT 52
3 MATERIALS AND METHODS
3.1 PATIENTS SELECTION 53
3.1.1 CONTROL GROUP 53
3.1.2 AMI GROUP 53
3.1.3 DIGOXTN TREATMENT GROUP 54
3.2 ANALYTIC METHODS 55
3.2.1 DETERJVONATION OF SERUM Magnesium 55 .
3.2.2 DETERMINATION OF SERUM ZmC 57
3.2.3 DETERMmATION OF SERUM COPPER 60
3.2.4 DETERMINATION OF SERUM SELENIUM 63
4 RESULTS 68
4.1 RESULTS OF EVALUATION OF ANALYTICAL
V METHODS 68
4.1.1 RESULTS OF METHODS EVALUATION OF
SERUM ZmC ASSAY 68
4.1.2 RESULTS OF METHODS EVALUATION OF
SERUM COPPER ASSAY 71
4.1.3 RESULTS OF METHODS EVALUATION OF
SERUM SELENIUM ASSAY 73
4.2 RESULTS OF PATIENTS STUDY 76
4.2.1 CONTROL GROUP 76
4.2.2 PATIENTS WITH ACUTE MYOCARDLAJ.
INFARCTION I DEMOGRAPHIC DATA 77
4.2.3 DIGOXIN GROUP 83
5 DISCUSSION 85
5.1 AMI PATENTS AND TRACE ELEMENT STATUS 85
5.2 MAGNESIUM AND ZEMC STATUS YN PATENTS
RECEIVESfG DIGOXEM TREATMENT 88
REFERENCE 91
“LIST OF TABLES 99
LIST OF TABLES 102
TABLES & FIGURES
vi 摘 要
本研究用原子吸收分光光度法检测了 94名急性心肌梗塞
(AMI)病人血清中锌，铜，硒和镁之浓度。入院时，24%
的病人血清镁降低；血清锌普遍降低且25%的病人血清锌明
显降低（〈8 ^imol/L)o结果表明低锌，高铜/锌比，肾功下降
和高龄是导致AMI后死亡率升高的因素。AMI病人血清硒
降低无统计学意义。血清镁降低在AMI病人中较多见，尽
管它对死亡率无影响，但对AMI病人适量补镁是必要的。
血清低锌影响AMI病人的机制可能与急性时象反应，营养
不良和梗塞程度的大小有关。
在另一组接受地高辛治疗的病菌人中，血清低镁和低锌的
发生率也很高，分别为20%和50%，提示对心肌梗塞病人和
长期接受药物治疗者有必要监测此两种元素之水平。 SUMMARY
Ninety-four AMI patients were studied for their serum zinc, copper, selenium and magnesium concentration. 24% of patients were hypomagnesemic on admission during AML
Zinc concentration was lowered during AMI and severe hypozincemia (less than 8 ^mol/L) were found in 25% of the patients. Low zinc level, high copper/zinc ratio, in addition to impaired renal function and advanced age were independent risk factors for mortality after AMI. There was no evidence for a lower selenium level among AMI patients. The results indicated that hypomagnesemia was not a rare event in AMI patients. Although it did not affect mortality, appropriate supplement with magnesium is indicated. Hypotheses were proposed for the effect of low zinc concentration on patient mortality including acute phase reaction, nutritional depletion and size of infarct.
Iin another group of patients on digoxin treatment, a high prevalence of hypomagnesemia and hypozincemia were also found. Monitoring of these elements for patients with chronic
1 found. Monitoring of these elements for patients with chronic illness and receiving long term medication is indicated.
2 1 LITERATURE REVIEW
1.1 MAGNESIUM
1.1.1 GENERAL FUNCTION OF MAGNESIUM
I. MAGNESIUM IS NOT A TRUE TRACE ELEMENT
It is the fourth most abundant cation in the body and the
second most abundant intracellular cation after potassium. As an
alkaline earth metal, magnesium has chemical properties distinct
from those of the transition metals. Compared with the transition
metals, magnesium interacts with other chemical species with a
strong electrostatic bonding component and a relative preference
for oxygen over nitrogen atoms. There are two major roles for
magnesium in biological systems: it can compete with calcium for
binding sites on proteins and membranes, and form chelates with
important intracellular anionic ligands, notably adenosine
triphosphate (ATP)[H.Sigel et al, 1990]
3 II. AN ESSENTLVL COFACTOR FOR ENZYMES
Magnesium ion (Mg^+) is pivotal in the transfer, storage and utilization of energy. It regulates and catalyzes some 300 enzyme systems. Magnesium acts as an essential cofactor for enzymes concerned with [Carfmkel.L, 1985]:
1) The Krebs cycle (i.e. pyruvate dehydrogenase, isocitrate dehydrogenase, oxoglutarate dehydrogenase, and succinyl-CoA synthetase).
2) glycolysis (i.e. hexokinase, phosphoglucose isomerase, phosphofmctokinase, phosphoglycerate kinase, phosphoglyceromutase, enolase, and pyruvate kinase)
3) oxidative metabolism
4) transmembrane transport of other cations such as calcium, potassium and sodium.
4 Magnesium can affect enzyme activity by:
(1) binding the active site of the enzyme (pyruvate kinase,
enolase)
(2) by ligand binding (ATP-requiring enzymes)
(3) by causing conformational changes during the catalytic
process (Na-K-ATPase)
(4) by promoting aggregation of multienzyme complexes(e.g.,
aldehyde dehydrogenase)
Magnesium's role in regulating cell membrane permeability
and transmembrane electrolyte flux are widely accepted. Reduced
extracellular magnesium concentrations result in an increase in
transmission across the myoneural junction. Magnesium also acts
to maintain a low resting concentration of intracellular calcium
ions; it modulates and controls cell Ca:+ entry and Ca"^ release
from the sarcoplasmic and endoplasmic reticulum, a necessary
prerequisite for the trigger function of calcium in several cellular
5 processes [W.E.C.Wacker, 1980].
Within the cell nucleus, Mg^+ regulates DNA synthesis. Large
numbers of Mg^+ ions are bound to the pentose-phosphate
backbone of DNA. By regulating DNA and RNA(i.e., RNA
synthetase, mRNA attachment to ribosomes, etc) synthesis and
structure, Mg^+ plays a vital role in regulating cell growth,
reproduction and membrane structure.[Cameron IL et al, 1989]
Magnesium influences the secretion of PTH by the parathyroid
glands, and severe hypomagnesemia may cause
hypoparathyrioidism.
1.1.2 ABSORPTION AND METABOLISM OF MAGNESIUM
!. Distribution in the human body
The adult human body (70 kg) contains 21 to 28 g of
magnesium (approximately 1 mol). About 70% total body
magnesium is present in the skeleton, 20% in skeletal muscle, 8%
in other cells, and about 2% in extracellular fluid [J.A.F.Rook et
al, 1962]
6 The main dietary sources of magnesium are vegetables containing chlorophyll, seafood, nuts, and grains contain appreciable amounts, whereas oils, fats. And sugars contain very little. In addition, drinking water, especially hard water, may be a major source of magnesium. The Recommended Dietary
Allowance (RDA) for magnesium is listed in Table 1-1.
About 30% of dietary magnesium is absorbed from the small intestin. However, this may vary widely because intestinal absorption is inversely related to magnesium intake. Magnesium absorption is affected by malabsorption syndromes, factors that affect transit time, calcium, phosphate, protein, lactose, or ingested alcohol. Vitamin D has not been shown to affect magnesium absorption [J. Behar, 1974:.
The major excretory pathway for absorbed magnesium is via the kidney. Magnesium homeostasis is achieved primarily via renal regulation. During periods of magnesium depletion, kidney magnesium excretion can be markedly reduced. The thick ascending loop of Henle is the major segment involved in control
7 of renal magnesium balance via both active and passive transport
mechanisms. The amount of magnesium being reabsorbed in the
distal tubule is load-dependent and has been shown to be closely
related to sodium, calcium, phosphate,and potassium reabsorption
and to the levels of various hormones (parathyroid hormone,
calcitonin, ADH). The major part of magnesium in plasma (about
55-60%) is present in the free ion form , and about 6-13 % is
complexed to phosphate and citrate; the remainder is bound to
protein, the majority being bound to albumin[Tietz et al, 1994].*
1.1.3 CLINICAL ASPECTS
I. Symptoms of hypomagnesimia
The symptoms of hypomagnesemia are very similar to those
of hypocalcaemia: impairment of neuromuscular function;
examples are hyperirritability, tetany, tremor, convulsions, muscle
weakness and electrocardiographic changes [M.J.Halpem，1985".
Magnesium deprivation has been associated with
cardiovascular disease through epidemiological evidence that
relates low magnesium intake to a high incidence of cardiac
8 deaths, particularly in soft-water areas where magnesium intakes are higher. Hypertension, myocardial infarction, cardiac dysrhythmias, coronary vasospasm, and premature atherosclerosis also have been linked to magnesium depletion [R.Whang et al,
1982].
II. Causes of hypomagnesemia
(1) Dietary insufficiency accompanied by intestinal malabsorption, severe vomiting, diarrhoea or other causes of intestinal loss. (2) Osmotic diuresis such as occurs in diabetes mellitus. Prolonged use ofdiuretic treatment especially when dietary intake has been marginal. (3) Cytotoxic drug therapy
such as cisplatinum which impairs renal tubular reabsorption of
magnesium. (4) Treatment with immunosuppressant drugs.
[MJ.Halpem, 1985]
Human magnesium deficiency, as indicated by reduced serum
magnesium concetration (hypomagnesemia), occurs with either
9 normal or reduced serum calcium concentrations.
Hypomagnesemia may be a secondary effect in hypocalcemic or calcium-deficient tetany. Yet a hypomagnesemic-normocalcemic tetany has been described that can be effectively treated with magnesium supplementation alone. During tetany, serum magnesium concentration of0.15 to 0.5 mmol/L accompanied by normal serum calcium and pH have been reported. There is evidence that tetany accompanied by hypocalcemia and hypomagnesemia may not be optimally treated with calcium administration alone. Decreased serum potassium concentrations
(hypokalemia) have also been found to accompany magnesium depletion. The occurrence of otherwise unexplained hypokalemia or hypocalcemia should suggest magnesium deficiency [Tietz et al, 1994].
Hypomagnesemia have been associated with chronic alcoholism, childhood malnutrition, lactation, malabsorption, acute pancreatitis, hypothyroidism, chronic glomerulonephritis,
aldosteronism, digitalis intoxication, and prolonged intravenous
feeding. Magnesium depletion occurs in condition that disrupt the
10 normal renal conservation of magnesium, for example, in patients
with renal tubular reabsorption defects and those taking
chlorothiazides, ammonium chloride, or mercurial diuretics for
congestive heart failure [Wong ET et al, 1982；.
Increased serum magnesium concentrations have been
observed in dehydration, severe diabetic acidosis, and Addison's
disease [Ryzen E et al, 1985]. Conditions that interfere with
glomerular filtration, for example, uremia, result in retention of
magnesium and hence elevation of serum concentrations.
Hypermagnesemia leads to an increase in the atrioventricular
conduction time of the electrocardiogram.[Cohen L et al, 1984"
1.1.4 METHODS OF DETERMINATION OF MAGNESIUM
I. Atomic Absorption Spectrophotometry (AAS)
Atomic absorption spectrophotometry is the preferred
technique for the determination of magnesium in biological
‘ specimens. This is due to its high sensitivity and specificity, small
sample size requirements. No interferences have been observed to
Mg detection from large concentrations of sodium, potassium,
11 calcium, and phosphorus, which are often present in biological smaples [W.Slavin, 1967:.
Graphite furnace atomic absorption spectrometry is an excellent method to provide sub-ng/ml minimum detection limits.
II Colormetric Methods
Many fluorometric (8-hydroxyquinoline) and colormetric
methods have been used for determining serum magnesium. The
colored calmagite-Mg complex is formed immediately upon
mixing calmagite reagent with serum and is stable for over 30
min. Serum magnesium is calculated by comparing the spectral
absorbance of the sample at 520 nm with magnesium iodate
calibrators. The method is simple and rapid.[Tietz et al, 1994]
12 1.2 zmc
1.2.1 GENERAL FUNCTION OF ZINC
Zinc is an essential trace element. Research has elucidated
many of its specific metabolic interactions since zinc deficiency
was first described in humans in the early 1960s. Zinc is an
essential component of over 200 metalloenzymes with a wide
range of functions eg. carbonic anhydrase, alkaline phosphate,
RNA，and DNA polymerases, thymidine kinase,
carboxypeptidases, and alcohol dehydrogenase [J.G.Reinhold et
al, 1976].
The zinc atoms are an integral, firmly bound part ofthe
metalloprotein molecule and are often involved in the active site;
they also contribute to the conformation and structural stability of
many metalloenzymes,[Tietz et al, 1994'
13 Zinc plays a role in various physiological functions including
growth, wound healing, skin integrity, functioning of male
reproductive organs and fertility, integrity of the immune system,
anti-inflammatory actions, protection against free radicals, and
functioning of taste and eyesight [R.L.Willson，1989；.
1.2.2 ABSORPTION AND METABOLISM OF ZINC
I. Distribution in the human body
Zinc is second to iron as the most abundant trace element in
the body. The body content in a 70-kg man is about 2.5 g. Zinc
are located mainly in muscle (60%), bone(30%), prostrate, semen,
liver, kidney and retina [MJJacksin, 1989]. The zinc content of
erythrocytes is about 10 times that of plasma because of their rich
content of carbonic anhydrase and other zinc-containing enzymes
[Tietz et al, 1994].
II. Intake of Zinc
Zinc is present in all protein-rich foods such as meat, fish, and
dairy products. The absorption of zinc is reduced in the presence
14 of phytates (inositol phosphates), cellulose, hemicellulose, and other dietary fibers from vegetables and cereal grains. Westem diets generally supply adults with 10 to 15 mg ZnAi. The RDA for zinc are listed in Table 1-1.
III. Absorption
Around 30% of ingested zinc is absorbed. Zinc absorption occurs mostly in the small intestine and especially in jejunum.
Zn2+ is absorbed possibly in the form of complexes with amino acids, citrate, etc. It is an active, energy dependent process. Zinc absorption is variable and is dependent upon a variety of factors.
Cereals, for example, are rich in zinc, but the absorption is poor due to the phytate ligands. Proteins and amino acids present in foods increase the absorption of zinc. The technologies used in food processing may lead to an increase or a decrease of zinc content in the foods psLW.Solomons, 1982；.
15 IV. Transportation
In the blood, zinc is distributed between red blood cells
(80%), plasma (17%), and white blood cells (3%). In plasma, 90%
of zinc is bound to albumin and 10% to a2-macr0gl0bulin with a
small amount associated with transferrin, glycoprotein,
transthyretine and free amino acids [W.R.Harris, 1983；.
V. Excretion
Zinc is mainly excreted via the feces. Pancreatic secretion
accounts for about 25% of total excretion. Biliary losses are small.
Loss through urine, sweat, milk, and sperm are minor.
1.2.3 CLINICAL ASPECT
I. General Causes ofzinc deficiency:
Nutritional zinc deficiency in humans is fairly prevalent
throughout the world. It occurs in both adults and children
through lack of dietary zinc. Old age, pregnancy, lactation, and
alcoholism are also associated with a higher incidence of poor
zinc nutrition.
16 II. Diseases Causing Zinc Deficiency:
1) hepatic cirrhosis
2) renal disease
3) bumed patients
4) neoplastic and inflammatory diseases
5) genetic disorder of zinc metabolism-acrodermatitis
enteropathica: This is a rare inherited disease which manifests itself in infancy as retarded growth, hypogonadism, dermatological and ophthalmic lesions, and gastrointestinal disturbances. The patients exhibit lowered plasma zinc concentrations. Untreated, the prognosis is poor, but oral zinc therapy (zinc sulfate) leads to complete remission.
6) Zinc is antagonized by cadmium, and zinc deficiency can be a consequence of chronic cadmium poisoning.
17 III. Iatrogenic Causes of Zinc Deficiency:
Zinc and other trace element deficiency is known to occur in
patients on synthetic diets therapies or total intravenous nutrition
(TPN) for a long term and causes a characteristic skin rash and
hair loss.
Administration of anabolic and metal-chelating drugs such as
corticosteroids and penicillamine [Tietz et al, 1994；.
IV. clinical features
In children, the rate of growth during rehabilitation from
famine has been clearly related to the dietary supply of
bioavailable zinc.
The clinical features ofzinc deficiency exist as a spectrum; on
one end, the symptoms may be mild or marginal, whereas on the
other end, the symptoms are severe. The main clinical
manifestations include skin injury, alopecia, poor wound healing,
anorexia, loss of senses, impaired development and function of
male reproductive organs, and immunodeficiencies.
18 1.2.4 ASSESSMENT OF THE BODY ZINC STATUS
Laboratory tests for assessing zinc nutrition can be classified
into two groups: those involving the analysis of zinc in a body
tissue or fluid and those testing a zinc-dependent function. Useful
tests in the first group include determination of the zinc content of
plasma or serum, blood cells, urine, and saliva. Functional tests
include measurement of activities of zinc-containing enzymes and
assessment of taste acuity. All of the above indices have been
shown to decrease with zinc deficiency. Other tests that are either
too complex for routine diagnostics or not well investigated
include skin and fingemail zinc determinations, Zn uptake by
erythrocytes, measurement of changes of plasma zinc content
during exercise, blood ethanol clearance, Zn retention and
tumover, and zinc balance.
Although the tests mentioned above have been shown to be
related to zinc depletion in animals and humans, no single test has
been proven to be a definitive indicator of zinc status. Test results
must be interpreted with caution because they may be confounded
19 by clinical conditions unrelated to the subjects zinc status per se.
Methological problems unique to trace element analysis have also
hampered the routine diagnosis of zinc deficiency [Tietz et al,
1994].
1.2.5 METHODS OF DETERMINATION OF ZINC
The analytical methods for zinc measurements in body fluids
include colorimetric and fluorimetric methods, AAS, neutron
activation analysis(NAA), and inductively coupled plasma mass
spectrometry (ICPMS).
I. Atomic absorption spectromitry (AAS)
Flame AAS is the analytically reliable and generally used test
for the determination of zinc in blood, serum, plasma, tears, and
tissues. Electothermal AAS is useful when lower levels of zinc
are to be determined.
20 1.3 COPPER
1.3.1 GENERAL FUNCTION OF COPPER
Copper is a component of a wide range of intracellular
metalloenzymes, including ceruloplasmin, cytochrome oxidase,
superoxide dismutase, dopamine-B-hydroxylase, ascorbate
oxidase, lysyl oxidase, and tyrosinase.
Iron needs copper to be absorbed .Copper deficiency leads to
anemia. Most of the copper in plasma is associated with the
specific copper-binding protein, caeruloplasmin. And
caeruloplasmin has a ferroxidase activity that oxidizes ferrous
iron to the ferric state prior to its binding by plasma transferrin.
[A.Mas et al, 1992]
1.3.2 ABSORPTION AND METABOLISM OF COPPER
1. Dietary intake
The copper content of foods vary according to the mineral
content soil in the area .from which the food is obtained.
Among the best food sources are liver, whole-grain products,
21 almonds, green leafy vegetables, shellfish, crustaceans, kidneys, nuts, molasses, cauliflower, avocados, organ meat,
eggs, poultry, and dried lgumes, whereas cows milk and dairy
products have low copper contents. The copper content in food
are also affected by the copper loss or contamination
throughout processing.
Many of the published values of the copper content of
foods are based on older data and give erroneously high
estimates. The RDAs for copper are shown in Table 1-1.
Althoug copper is present in most foodstuffs, there is
evidence that not all modem diets contain sufficient copper,
especially when large amounts of refined carbohydrate are
consumed.
II. Absorption
About 50% to 80% of the average daily dietary copper of
around 25 ^imol (1.5 mg) is absorbed from the stomach and the
small intestine. The mechanisms by which copper is absorbed
and transported by the intestine are unclear. The exact role of
22 metallothionein in the intestinal absorption of copper is not well understood although zinc diminishes copper absorption by
increased production of metallothionein in mucosal cells which then binds copper preferentially. Thus intestinal
metallothionein may have some function in maintaining copper
homeostasis. [T.U.Hoogenraad et al, 1983；
Factors affecting copper absorption include gender (women
absorbing a greater percentage than men), the amount infested,
chemical form, and certain dietary constituents including other
trace elements, sulfate, various amino acids, fiber, and phytates.
Copper absorption may be impaired in patients with diffuse
diseases affecting the small bowel such as sprue,
lymphosarcoma, and scleroderma.
IIL Transportation
Absorbed copper is rapidly transported in portal blood
bound to albumin or histidine to the liver where it is stored,
mostly as metallothionine-like cuproproteins. Copper is
exported to peripherial tissues mainly as ceruloplasmin which
23 is a multifunctional cuproprotein that accounts for over 95% of
the total copper in plasma and, to a lesser extent, albumin,
transcuprein, and amino acid. The movement of copper within
the cell and its incorporation into cuproproteins is regulated by
both glutathione and metallothionine.[Frieden, 1980:
IV�Distribution
The adult human body contains between 80 and 150 mg of
copper which is present in all metabolically active tissues. The
highest concentrations are found in liver (30-50 g/g dry tissue)
and in kidney, with significant amounts in heart, brain. Muscle
and bone copper concentrations are low but constitute about
50% of the total-body copper because of their large mass.
[D.H.Hamer, 1986]
V. Homeostasis
Copper is excreted primarily into the gut, from unabsorbed
‘ dietary copper, and from biliary and gastrointestinal secretions.
Biliary copper excretion ranges from 0.5 to 1.3 mg/d.
Gastrointestinal sources include copper from gastric juice and
24 from desquamated mucosal cells. Copper losses in the urine
and sweat amount to less than 3% of dietary intake. Menstrual
losses of copper apparently are minor: 0.1 to 0.8 mg per period.
1.3.3 CLINICAL ASPECT
1. Human Copper Deficiency
Copper deficiencies are relatively unknown. Low blood
levels have been observed in children with copper or iron-
deficiency anemia, prematurity, malnutrition, malabsorption,
chronic diarrhea, hyperalimentation, edema and prolonged
feeding with total milk diets. Symptoms of a deficiency include
general weakness, impaired respiration, osteoporosis and
various bone and joint abnormalities, decreased pigmentation
of the skin and general pallor and skin sores, and possible
neurological abnormalities (hypotonia, apnea, psychomotor
retardation). Inadequate intake can lead to neutropenia and
hypochromic anemia. [E Frieden et al, 1983"
Human copper deficiency has also been associated with
zinc therapy for sickle cell disease and treatment with copper-
25 chelating agents like penicillamine.[Tietz et al, 1994]
II. Menkes Syndrome
This is an X-linked genetic disorder characterized by focal
cerebral and cerebeller degeneration, retardation of growth, and
kinky or steely hair. It is an extreme form of copper deficiency
with defect in copper transport and storage.
III. Wilsons Disease
A rare autosomal recessive inherited disease with abnormal
copper metabolism, which brings about excess copper retention
in the liver, brain, kidney, and comeas of the eye. It is
characterized by cirrhosis of the liver, irreversible dseases in
the central nervous system, and comeal degradation.
IV. Copper Toxicity
Copper is nontoxic to humans in small amounts; up to 35
mg daily for an adult is safe. Conditions that show high copper
blood levels include oral contraceptives, stuttering, autism,
childhood hyperactivity, toxemia of pregnancy, premenstrual
26 tension, depression, insomnia, senility, premature baldness, tinnitus, facial pigmentation, painful joints, functional hypoglycemia, cirrhosis of the liver, hypoproteinemia, niacin deficiency, infections, heart attack, leukemia, and mental illness. It can occur subsequent to ingestion of copper- contaminated solutions, the use of copper-containing intrauterine devices, the use of copper salts in animals feeds, and exposure to copper-containing fungicides. Systemic toxic effects include hemolysis, hepatic necrosis, gastrointestinal bleeding, oliguria, azotemia, hemoglobinuria, hematuria, proteinuria, tachycardia, convulsions coma, and death. [Linder et al, 1991]
Because the liver is an important organ for copper storage and homeostatic regulation, abnormal copper metabolism is seen in liver disease. Elevated serum copper concentrations are seen in portal cirrhosis, biliary tract disease, and hepatitis, probably because excess copper that would normally be excreted in the bile is retained in the circulation. Hypocupremia has been observed in hemolytic jaundice, hemochromatosis,
27 and some types of hepatic cirrhosis because of the inability of
the damaged liver to synthesize ceruloplasmin. [Tietz et al,
1994]
1.3.4 LABORATORY ASSESSMENT OF COPPER STATUS
As with zinc, the assessment of copper nutrition in adult
humans has not been perfected. There are, however, a number
of indices that are useful in the diagnosis of human copper
deficiency: Serum or Plasma Copper Concentrations,
Ceruloplasmin, Superoxidase Dismutase and Cytochrome c
Oxidase Activities, erythrocyte and leukocyte copper content,
skin morphology, and measurements of copper retention and
turnover.
28 Copper is associated with two main plasma proteins,
albumin and caeruloplasmin. The major copper transport
protein may be albumin. Concentrations of caeruloplasmin is
unrelated to dietary copper. [Linder et al, 1981:
1.3.5 METHODS FOR THE DETERMINATION OF COPPER
Atomic absorption spectrophotometry after direct dilution
is used frequently to quantitatively estimate serum copper.
Photometric methods are also available, such as those using
diethydithiocarbamate, bis-cyclohexanone, oxaldihydrazone
(cuprizone), and bathocuproine as chromogens. Hemolysis is
not a great concern for copper determinations because
concentrations of copper in plasma and erythrocytes are nearly
equal. Reference intervals for serum copper have been
compiled and are higher in pregnancy, 118 to 302 g/dL (18.5-
47.4^imolA.); in children 6 to 12 years of age, 80 to 190 gAlL
(12.6-29.9 ^imol/L); and in women, 80 to 155 g/dL (12.6-24.4
^mol/L) than in men, 70 to 140 g/dL (11.0-22.0 ^unoLO^); and
in infants, 20 to 70 g/dL (3.1-11.0 ^imol/L),
29 Serum ceruloplasmin can be determined by a photometric oxidase reaction, by immunonephelometry, or by radial immunodiffusion. The reference interval of 180 to 450 mg/L varies slightly depending upon the method used.
30 1.4 SELENIUM
1.4.1 GENERAL FUNCTION OF SELENIUM
Selenium is an essential element for humans. It is a cofactor
of glutathione peroxidase. This enzyme protects membranes
from damaging effects of peroxides (hydrogen peroxide and
lipid hydroperoxide) by decomposing these oxidizing
compounds. It is part of the cellular antioxidant defense system
against free radicals. It complexes with heavy metals and
protects against cadmium and mercury toxicity.
It also plays a role in electron transfer functions. It also may
affect drug-metabolizing enzymes and is involved in the
metabolism of thyroid hormones.
1.4.2 ABSORPTION AND METABOLISM OF SELENIUM
I. Dietary Intake
Selenium is present in foods as selenomethionine or
selenocystine; selenomethionine is derived from plants and
selenocysteine from animal sources. Good sources of selenium
31 include seafood, organ meats, muscle meats, and whole grains.
Fruits and vegetables are poor sources. The selenium content of
foods varies widely from one region to another and is directly related to food protein content. The dietary intakes in the united states often tend to fall within the range of 83-129 p,g/day.
Much lower selenium intakes are found in New Zealand (28-
32^imol/L) and Finland (30-50 ^imol/L). Most extremely low value come from the Keshan disease endemic area of China (7^ g /d). On the other hand, high dietary intakes of selenium occur in areas with naturally seleniferous soils such as the chronic selenosis area in China (4.9 mg/day). [R.A.Zingaro et ai, 1974]
IL Absorption
Selenium is efficiently absorbed from the gastrointestinal tract (varying from 35-85%) and is highest in the duodenum and absorption is apparently not regulated. Little is known about the mechanisms of absorption, it may be through a wide variety of mechanisms, e.g. selenomethionine is absorbed by the same mechanism as methionine.
32 III. Distribution
Selenium may be transported from the gut to the liver mainly on VLDL and LDL [Cavalieri, 1980] and achieves its highest tissue concentrations in red cells, liver, spleen, heart, nails, and tooth enamel. It also concentrates in the testes and sperm, which are also more refractory to dietary deprivation.
Mason, 1988] Selenium is present in tissues in two major forms: selenocysteine and selenomethionine. Selenomethionine can not be sysnthesized by the diet. It can replace methionine in a variety of proteins. Selenomethionine is considered as an unregulated storage compartment for selenium. When the dietary selenium supply is interrupted, this pool tums over and supplies selenium to the organism. Selenocysteine is present in selenoproteins eg. glutathione peroxidase, throxine-5�- deiodinase, and selenoprotein p. Other forms of selenium may be present tissues but have not been fully characterized.[Tietz et al, 1994]
33 IV. Excretion
Selenium is normally excreted in the urine and lungs and its
presence in the feces is an indication of improper
absorption.Selenium is present in bile in low concentration.
1.4.3 CLINICAL ASPECT
I. Keshan disease
This is an endemic cardiomyopathy that affects primarily
children and woman of childbearing age in certain areas of
China, has been associated with selenium deficiency.
II. Kashin-Beck disease
It is an endemic osteoarthritis that occurs during adolescent
and preadolescent years, is another disease linked to low
selenium status in China. The exact cause of this disease is
unknown and other factors such as mineral imbalance,
mycotoxins in grain, or organic contaminants in drinking water
may be involved.
34 III. Cardiomyopathy Cauesed by Selenium Deficiency
Epidemiological studies have related cardiomyopathy with
selenium deficiency from the consumption of low selenium
content cereals. [X.S.Chen et al, 1980] Cardiomyopathy and
low glutathione peroxidase activities have also been reported in
patients on TPN who were not supplemented with selenium.
The anticarcinogenic effects of selenium was supported by
experiments with rodent models.
IV. Toxicity
Signs of chronic selenosis include hair and nails, skin
lesions, tooth decay, malformed hooves, lameness, emaciation
and nervous system abnormalities. Toxicity from
administration of selenium compounds for the treatment has
been reported (M.M.W.R., March 30, 1984). Acute selenosis
produces severe symptoms including nausea, vomiting, hair
• loss, nail changes, irritability, fatigue, and peripheral
neuropathy and may die in a few hours.
35 1.4.4 ASSESSMENT
The determinations of urinary and blood selenium are widely used as indicators of selenium status. Plasma or serum selenium concentrations are sensitive to dietary intake. Red cells were found with high concentration of selenium (0.23-
0.36 ^ig/ml of cells), and hemolysis can alter the serum levels.
Most of the selenium in serum is in the form of seleocystein in proteins. Therefore hypoprotienemia can lead to falsely lowered concentration of serum selenium. In China, a correlation was found between hair selenium concentrations and blood concentrations. A functional test for assessing selenium nutrition is to measure glutathine peroxidase in red ‘ cells. [Diplock A.T., 1993] Blood and tissue selenium contents vary with the selenium status of the particular geographical area. Reported selenium concentrations in specimens from healthy adults are listed in tablel-3. Unfortunately, local data are not available.
36 1.4.5 METHODS OF DETERMEVATION OF SELENDJM
L Methods
Seven methods were employed for quantitating selenium in
biological specimens: neutron activation analysis fMAA)
Spectrofluorometry, electrothermal ASS, hydride generation
AAS, isotope dilution mass spectrometry ^MS), XRF
spectrometry, inductively coupled plasma (ICP).
Recommended methods for determining selenium in biological
specimens are flameless ASS.
II. Analytic Problems
一
There is great variability in results among different
laboratories. The main reasons for inaccurate and imprecise
results include sampling errors, contamination, losses during
handling, pretreatment, decomposition and steps in processing.
The organo-selenium forms are likely to be volatile and
therefore can be lost in step ofpyrolysis.
37 III Electrothermal (flameless) atomic absorption
Spectrophotometry
The conventional flame AAS is not sensitive enough for
Selenium determination. Flameless AAS ofers high
sensitivity, although interferences increase. Advantages ofthe
flameless AAS are increased detection limits, requirement of
low sample volumes and the simplification of sample
pretreatment.
1.5 INTRODUCTION TO ATOMIC ABSORPTION
SPECTROPHOTOMETRY
1.5.1 PRINCIPLE OF AAS
The atom is made up of a nucleus surrounded by electrons.
The lowest energy is known as unexcited or ground state which is
the most stable electronic configuration of an atom. If energy of
the right magnitude is applied to an atom, the energy will be
absorbed by the atom and an outer electron will be promoted to a
less stable configuration or excited state. As this state is unstable,
38 the electron will retum to its initial stable orbital position (decay
process), and radiant energy will be emitted. The wavelength of
light emitted is a unique property of each individual element.
(Figure 1-1 & Figure 1-2)
Absorbance (A) is calculated as the formula below:
A 二 log (Io/I)
Io is the initial intensity of the light at the resonance
wavelenghth，I is the reduced intensity after the atomic absorption
process in the sample cell.
1.5.2 INSTRUMENTATION
^ I. FLAME AAS
Every absorption spectrometer must have components which
fulfil three basic requirements: 1) a light source; 2) a sample cell;.
and 3) a means of specific light measurement. (Figure 1-3)
The two common line sources used in atomic absorption are
the hollow cathode lamp and electrodeless discharge lamp. The
atomizer is a key component of the AAS instrument which
39 temperatures to bum off any sample residue which may
remain in the fumace.
6 Cool down - allows the flimace to retum to near
ambient temperature prior to the introduction of the
next sample.
42 2 INTRODUCTION
2.1 TRACE ELEMENT DEFICIENCY IN HOSPITAL PATIENT
Hospital patients are prone to develop malnutrition. Those
with severe illness and at the extremes of age are particularly at
risk. Unbalanced intake and loss from the body is the major
reason for malnutrition. As trace elements are mostly water
soluble, deficiency will develop early after a negative balance is
established. It is in contrast to other predominantly lipid-soluble
nutrients such as lipid -soluble vitamins.
Rammohan et al (1989) evaluated the nutrient intake in
hospitalized elderly patients of21 hospitals in the United States.
They found that more than 60% of the patients had inadequate
intake of a form of nutrient. Insufficient zinc and magnesium
intake was found in 90% of patients older than 65 years.
Therefore it is expected that deficiency of these trace
elements are common among hospitalized patients.
43 2.2 MICRONUTRIENT DEFICIENCY AMONG PATIENTS
WITH ACUTE MYOCARDIAL INFARCTION
Heart diseases are a cause of considerable mortality in
westem countries. It is well established that atherosclerosis is a
multifactorial disease. Nutrition is known as one of the most
important causes.
2.2.1 MAGNESIUM DEFICIENCY
The pathogenesis of low serum magnesium concentrations
in patients with MI has not been fully elucidated. The bulk of
experience from clinical studies mirrors the findings of animal
experimentation, in which serum magnesium concentrations
decreased within 1 hour following coronary occlusion and
returned to normal by 24 to 48 hours. This decrease has been
attributed to a number of factors, including hemodilution, renal
• excretion, or increased intracellular influx with binding to free
fatty acids. Careful recent work in patients with MI has
excluded the first 2 hypotheses. The remaining hypothesis,
44 which attributes low serum magnesium concentrations in patients with MI to an intracellular magnesium shift mediatd by catecholamine-induced lipomobilization, is being supported by a large number of laboratory and clinical investigations.
There were also evidence to support an effect due to the occurrence of lipolysis, release of long-chain free fatty acids, and hypomagnesemia during acute MI, during endogenous rise of catecholamine, during infusion of adrenaline, or other stress factors. The release of free fatty acids and hypomagnesemia occur simultaneously, and both have been attributed to catecholamine release.
A large clinical trial ('Limit-2' program) studied the beneficial effect of IV magnesium treatment in patient suffered from AMI. A positive result in term of decreased acute & long term mortality was evident [Woods et al, 1992]. Furthermore, a low serum magnesium was found in high percentage ofpatients with AMI on admission.
45 Hypomagnesium was found to increase the risk of supraventricular and ventricular arrhythmia during AMI. And these arrhythmias are major causes of mortality and morbidity in AMI. Therefore magnesium level on admission could be a major prognostic factor in predicting outcome in these patients and patients found to have a low semm magnesium should be treated with supplement.
However, there are a large difference in the prevalence of magnesium deficiency in different part of the world. A high percentage of hypomagnesemia was found among patients with chest pain by Salem et al (1996) in the United States. The prevalence increased further to 56% among those receiving diuresis.
On the other hand, Ellis et al (1988) has not observed a significantly lower magnesium concentration among patients with AMI in a study in Australia. Therefore it is important to establish the prevalence of magnesium among local patients
46 with AMI in order to determine the value of IV magnesium
therapy during the acute phase and the replacement of
magnesium during hospital period.
Furthermore, we also studied a group of patients who were
on digoxin treatment. Digoxin is a potentially toxic drug and
monitoring of its serum level does not reflect clinical toxicity in
many occasions. Deficiency of plasma electrolytes, especially
plasma Mg leads to an increased susceptability to digoxin
toxicity. [Douban S, 1996]
2.2.2 Change ofZinc and Copper in AMI
It has been suggested that the injury induced by reperfusion
of the ischemic myocardium could result partially from the
cytotoxic effects of oxygen free radicals. Zinc and copper are
involved in several of the reactions in the protection from free
radical damage.
I. They are components of several metalloenzymes with redox
47 capacity. This redox capacity gives them an anti-oxidizing and
anti-reactive oxygen radical action.
11. Zinc and Copper are also components of a protein:
metallothione (MT).
MT is characterized mainly by a high cysteine content
(30%) and metal-binding capacity (7 atom Zn/mol) and 9 atom
Cu/mol). The synthesis ofMT is induced by metals such as Cd,
Zn and Cu, and also indirectly by non-metallic compounds as
glucocorticoids and hormones.
Variation in trace element can occur in several pathological conditions. Precisely, different alterations of Zn, Cu serum
concentrations are produced during acute myocardial infarction
(AMI), including a decrease of Zn and an increase in Cu serum
levels, which take place within the first hours of AMI onset. A
decrease in serum zinc levels has been reported after acute
myocardial infarction with a concomitant increase in serum
48 copper. These variations have been related to the classic enzymatic markers of this pathology (AST, LDH, CK-MB).
[Speich M，1988]
In an AMI study, increased lipid peroxidation after induced
AMI was found in rats treated with a zinc deficient diet
[Condray et al, 1993]. There was also a large infarct size among those zinc deficient rats. The effect of zinc supplement on myocardial infarct size in dogs was studied [Lal a, 1991]. Zinc supplement appeared to be potentially prophylactic for limiting the size of myocardial infact in the dogs.
Furthermore, a zinc study [Vilanova et al, 1997] showed that a low serum zinc concentration on admission was associated with a high mortality in AMI patients.
Zinc is an important constituent of various metalloenzymes that control the synthesis of nucleic acids and proteins.
Therefore, the fall in serum zinc levels could be due to the large
49 amount of zinc needed by the myocardial tissue to participate in
the process of repair. However, zinc variations could also be
explained by stress and a preexisting deficient state. Therefore
it is important to determine if low plasma zinc is associated
with prognosis after AMI in local patients.
The suggestion that subclinical copper depletion contributes
to an increased risk of coronary heart disease through instability
of heart rhythm and hyperlipidemia is well supported
experimentally and epidemiologically. Experimental animals
fed copper-deficient diets and human subjects fed diets believed
to be marginal in copper have exhibited many symptoms
closely related to coronary heart disease. These include
abnormal electrocardiograms, hypercholesterolemia, glucose
intolerance, and hypertension. [Tietz et al, 1994"
2.2.3 SELENIUM DEFICIENCY
Substantial and growing-evidence from a variety of sources
support the hypothesis that oxidation of low-density lipoprotein
50 may have a role in the etiology of atherosclerosis. Antioxidant may protect against free radical mediated carcinogenesis. The antioxidant properties of selenium, a constituent of glutathione peroxidase, are well known, and the hypothesis that this element may reduce cardiovascular disease has recently been reviewed. Although low plasma selenium has not been directly linked with susceptibility of low-density lipoprotein to oxidation, glutathione peroxidase has a major role in reducing peroxidation processes. Low plasma selenium has also been associated with increased platelet aggregability, higher production of thromboxane A2, and decreased production of prostcyclin, all of which may potentially be linked to cardiovascular disease. In Finland, a low-selenium region, persons with levels <45 [ig/L had an increased risk of cardiovascular death and myocardial infarction (MI). However, subsequent studies have yielded conflicting results.
In a multicenter study examining the association between
51 3 MATERLVLS AND METHODS
3.1 PATIENTS SELECTION
3.1.1 CONTROL GROUP
63 patients attending out-patient clinic, who had no
history of MI and not receiving digoxin or diuretics
treatment were recruited as control. The control group served
to verify the already established reference intervals of serum
zinc, copper and magnesium. It was taken for comparison of
serum selenium with patients in AMI group.
3.1.2 AMI GROUP
Samples were collected from one study on evaluation of
change in cardiac enzymes in Chinese patients. Blood was
collected within 12 hours after presentation of chest pain. All
serum samples were frozen at -70^C before analysis. Totally
134 samples were analyzed.
53 Diagnosis of AMI was based on combination of
chest pain and standard ECG criteria or enzymatic evidence
ofmyocardial infarction. By this way, 94 patients were
ascertained suffering from AMI. The other patients were
classified having other unstable angina or arrhythmia.
Mortality ofthe group of patients were followed up by
hospital record and hospital computer registry.
3.1.3 DIGOXIN TREATMENT GROUP
101 patients requesting for a serum digoxin concentration
monitoring were studied. An aliquot of serum was saved for
measurement of magnesium and zinc. Serum copper was
also measured in 21 ofthem.
54 3.2 ANALYTIC METHODS
3.2.1 DETERMEVATION OF SERUM MAGNESIUM
The determination of serum Magnesium was performed
on Vitros 250 instrument (Johnson & Johnson, New
York,USA) by a dry chemistry method.
I. Materials
Reagent: Vitros Mg Slides
Reactive ingredients on the slide are 1,2-bis (o-
aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (calcium
chelator) and 1,5 -bis (2-hydroxy-3,5 -dichlorophenyl)-3 _
cyanoformazan (dye). Other ingredients include pigment , •
binders, buffer, dye solubilizer, surfactants, cross-linking
.agent.
55 II. Principle of the Assay
The Mg Slide is a dry, multilayered, analytical element coated on a polyester support. A 10 ^iL drop of patient sample is deposited on the slide and is evenly distributed by the spreading layer to the underlying layers. Magnesium
(both free and protein-bound) from the sample then reacts with the formazan dye derivative in the reagent layer; the high magnesium affinity of the dye dissociates magnesium from binding proteins. The resulting magnesium-dye complex causes a shift in the dye absorption maximum from
540 nm to 630 nm. The amount of dye complex formed is proportional to the magnesium concentration present in the sample and is measured by reflection density.
III. Assay Protocol for Magnesium
Test Type : Colorimetric
Wavelength: 630 nm
Assay Time: Approximately 5 minutes
56 Temperature: 37¾
Reaction Sequence
Mg+2 + Ca+2 chelator Mg+: + Ca^^-chelator complex
Mg+2 + formazan dye derivative pH 9.75
1^ Mg -dye complex
3.2.2 DETERMINATION OF SERUM ZINC
CONCENTRATION
The determination of serum zinc concentration was
completed on the AAS 300, Perkin Elmer Spectrophotometer
(New Jersey, USA).
57 I. Materials
Reagent
Nitric acid : Merck KgaA, Germany, Suprapur® 64271
Trichloro-acetic acid: Lot 35H0783, SIGMA (St. Louis)
Zinc stock standard (15295 umol/O： Perkin Elmer
Zinc QC: Perkin Elmer Atomic Spectroscopy Standard, Lot
No 5-94ZN
Working standard:
Std3 30.59 umoLT. (dilute stock standard x500 with
2% nitric acid)
Std2 20.39 umol/L (5 niL 2% nitric acid + 10 mL
standard 3)
Stdl 10.20 umoVL (10 mL 2% nitric acid + 5 mL
standard 3)
58 Working Aqueous QC (Dilute aqueous 1530 ^imol/L
QC X 100 with 2% nitric acid)
II. Sample Preparation
Mix 0.5 ml standard /QC/ patient sample with 2 ml 5%
TCA in a 5 ml-size pp tube. Centrifuge at 3,000 rpm for 10 minutes. Transfer the supernatant to another tube for analysis.
III Instrument Setting
wavelength 213.9 rnn
slit width 0.7 nm
read time 2.5 s
read delay 0 s
signal measurement time average
flame type air/C2H2
59 oxidant flow 10.00 L/min
fuel flow 3.00 L/min
calibration equation zero intercept: linear
3.2.3 DETERMINATION OF SERUM COPPER
The determination of serum copper was completed on the
AAS 300，Perkin Elmer Spectophotometer.
1. Materials
Reagent
Nitric acid : Merck KgaA, Germany, Suprapur® 64271
Trichloro-acetic acid: SIGMA, Lot 35H0783
Copper stock standard (15734 umol/):Perkin Elmer
Copper QC: Perkin Elmer Atomic Spectroscopy Standard,
Lot No 5-9CU
60 Working standard:
Std3 31.47 umol/L (dilute stock standard x500 with
2% nitric acid)
Std2 15.74 umoVL (5 mL 2% nitric acid + 10 mL
standard3 )
Stdl 7.87 umolA. (15 mL 2% nitric acid + 5 mL
standard3 )
Working Aqueous QC (Dilute aqueous 1573 umolA.
QC X 100 with 2% nitric acid)
n. Sample Preparation
Mix 0.5 ml standard /QC/ patient sample with 2 ml 5%
TCA in a 5 ml-size pp tube. Centrifuge at 3,000 rpm for 10 minutes. Transfer the supernatant to another tube for analysis.
61 III. Instrument Setting wavelength 324.8 nm
slit width 0.7 nm
read time 2.5 s
read delay 0 s
signal measurement time average
flame type air/C2H2
oxidant flow 10.00 L/min
fuel flow 3.00 L/min
calibration equation zero intercept: linear
replicates 2
62 3.2.4 DETERMEVATION OF SERUM SELENIUM
The graphite ftimace atomic absorption
spectrophotometer used in the measurement of selenium is
Perkin Elmer SCVtMA 6000 Spectrophotometer.
1. Materials
Reagent:
Nitric acid : Merck KgaA, Germany, Suprapur® 64271
Triton X-100 SIGMA Lot No 27F-0013
Modifier (0.5 mL 1% pd0^O3)2 + 0.3 mL 1%
MgO^fO3)2 + 9.2 mL D.W.)
Selenium stock standard (12665 umol/): Perkin Elmer
Selenium QC: Perkin Ehner Atomic Spectroscopy Standard,
Lot No 5-22SE
63 Working Aqueous QC reagent A: was prepared by diluting 1 mL stock(1266 mol/1) to 200 mL with 2% nitric acid. The selenium concentration of A was 6.332 umoL^.
(i) (High, 1.055 umol/L) 1 mL of(A) + 5 mL of2% nitric
acid
(ii) (Medium, 0.352 umolA.) 1 mL of (i) + 2 mL of 2%
nitric acid
(iii) (Low, 0.088umoL^) mL of (ii) + 3mL of2% nitric acid
U. Sample Preparation:
Mix 300 uL 0.25% Triton X-100 with 200 uL sample/QC in eppendorf tube. Centrifuge at 13.000 r.p.m. for 5 minutes. Transfer the &upematant to sample cup for analysis.
64 III. Instrument Setting
The parameters were modified from the
recommendation of the manufacturer.
Wavelength 196 nm
Read time 5 s
Read delay 0 s
Signal measurement peak area
Sample volume 20 uL
Modifier volume 10 uL
Calibration equation zero intercept, linear
Replicate . 2
65 Pipetting steps pipette diluent + modifier
+ sample/std
Pipetting speed 50%
Fumace conditions ( C: compressed air, A: argon)
"Sl^"""Temp~~~Ramp~~Hold~~Internal flow~~G^ Read~~ � C Time Time 1 no 1^ ^~~^ c 2 130 10s 40s 250 C 3 1200 15s 20s 250 A 4 1900 Os 5s 0 A X 5 2400 ls 5s 250 A
IV. Contamination Preventing
Due to the low serum selenium concentration, it is the
analytes that would be most likely influenced by any
contamination from the environment. Trace of selenium
present in the sample tube could lead to a major effect on
the observed results. Therefore, prevention of any selenium
in the sample tube is examined in a prior study. Ten new
66 sample tubes that were used in routine blood collection were checked. 5 ml deionized double distilled water was injected through a new blood syringe into each of the sample tubes.
The tubes were vortexed ovemight before the injected water
sample was analyzed for selenium.
All the ten samples used in evaluation of contamination
showed a selenium concentration less than 0.008 |imol/L
which are not different from the lower detection limit.
67 4 RESULTS
4.1 RESULTS OF EVALUATION OF ANALYTICAL
METHODS
4.1.1 RESULTS OF METHODS EVALUATION OF SERUM
ZINC ASSAY
I. WITHlN-BATCH PRECISION (Table 4-1)
Three samples of different levels were analyzed for 20
replicates.
Low Median High
N 20 20 20
Mean (^imoLO.) 12.29 21.39 33.03
.SD (^imoUL) 0.279 0.252 0.508
CV 2.3% ， 1.2% 1.5%
68 IIBETWEEN-BATCH PRECISION (TABLE 4-2)
Aqueou QC Semm QC
N 14 14
Mean(jimoVL) 16.13 22.59
SD (|imoUL) 0.59 0.77
CV 3.6% 3.4%
III. ACCURACY (TABLE 4-3)
10 TEQAS samples were analyzed, accuracy ranged
from 100.6 — 109.5%. (TEQAS: external quality control
sample from UKEQAS trace elements scheme)
IV. RECOVERY
For samples with large volume of standard addition:
(TABLE 4-4)
Five samples with 6.12 [xmoUL zinc added, recovery
ranged from 109.3-145.1%.
69 Five samples with 10.20 ^imol/L zinc added, recovery
ranged from 79.2-115.1%.
For samples with small volume of standard addition:
(TABLE 4-4)
Five samples with 5.53 ^imol/L zinc added, recovery
ranged from 92.4-105.6%.
Five samples with 10.67 ^mol/L zinc added, recovery
rangedfrom91.0-94.8%.
V. LINEARITY AND DETECTION LIMITS
(TABLE 4-6 & FIGURE 4-1)
Zinc assay was performed on a serial dilution of a high
concentration of standard to check the linearity of the
assay. The linearity range was between 9.4 and 18.4 [i
mol/L.
The lowest detection limit was 1.6 ^imolyT..
70 4.1.2 RESULTS OF METHODS EVALUATION OF SERUM
COPPER ASSAY
L WITfflN-BATCH PRECISION (Table 4-7)
Three samples of different levels were analyzed for 20
replicates.
Low Median High
N 20 20 20
Mean (^mol/L) 8.99 20.31 36.11
SD (^moVL) 0.108 0.353 0.325
CV 1.2% 1.7% 0.9%
71 II. BETWEEN-BATCH PRECISION (TABLE 4-8)
Aqueou QC Serum QC
N 14 14
Mean (^imol/L) 16.13 19.51 ^mol/L
SD (^imol/L) 0.59 0.74 |imol/T.
CV 3.6% 3.7%
III. ACCURACY (TABLE 4-9)
10 TEQAS samples were analyzed, accuracy ranged
from 92.2 — 97.0%.
IV. RECOVERY (TABLE 4-10)
Five samples with 6.29 ^imol/L copper added, recovery
ranged from 93.8-100.21%.
Five samples with 10.49 ^imol/L copper added,
recovery ranged from 101.6-104.3%
72 IV. LINEARITY AND DETECTION LEVIITS
(TABLE 4-11 & FIGURE 4-2)
Copper assay was performed on a serial dilution of a
high concentration of standard to check the linearity of the
assay. The linearity range was between 2.0 and 36.0 ^i
mol/L.
The lowest detection limit was 2.0 ^imol/L.
4.1.3 RESULTS OF METHODS EVALUATION OF SERUM
SELENIUM ASSAY
L PRECISION
Precision study consisted of within-batch precision and
between-batch precision. Within- batch precision (Table 4-
12) was performed by analyzing 20 replicates of low,
medium and high level control sera. The controls used in this
study were prepared by mixing patient samples. The low and
high levels were obtained by dilution or by addition of
73 selenium.
Between - batch precision (Table 4-13)was performed by analyzing one set of the low, medium and high concentration of QC materials and a commercial QC serum
(SERONORM™ Trace Elements Serum, Batch no. 311089).
The mean, SD and CV were then calculated and tabulated.
II RECOVERY
The basal serum sample was prepared and divided into five aliquots. Five different volumes of selenium standard
(0.672 ^imol/l) was added to each aliquot of basal sample.
The selenium concentration of each sample were determined in triplicate. The average of each was used for the calculation. Recovery ranged from 94% to 103%.
III LINEARITY AND DETECTION LIMITS
(FIGURE 4-3)
Selenium assay was performed on a serial dilution of a
74 high concentration of standard to check the linearity of the assay. The linearity range was between 0.021 and 15.0 ^ molyT^. The lowest detection limit was 0.021 ^mol/L.
75 4.2 RESULTS OF PATIENTS STUDY
4.2.1 CONTROL GROUP (Summary in Table 4-16)
I. SERUM MAGNESIUM
40 samples from this group were randomly selected for
analysis. The mean was 0.9 mmol/L. The minimal level was
0.57 mmol/L and the maximal level was 1.15 mmol/L.
II. SERUM ZINC
All 63 samples from this group were analyzed. The mean
was 11.3 ^imol/L. The minimal level was 4.0 ^imol/L and the
maximal level was 17.16 ^mol/L.
III SERUM COPPER
62 samples from this group were randomly selected for
analysis. The mean was 18.0 ^imol/L. The minimal level was
9.81 [imolfL and the maximal level was 28.2 |imol/L.
76 IV SERUM SELENIUM
57 samples from this group were randomly selected for
analysis. The mean was 0.87^mol/L. The minimal level was
0.52 ^imol/L and the maximal level was 1.65 ^imol/L.
4.2.2 PATIENTS WITH ACUTE MYOCARDIAL
INFARCTION
1. DEMOGRAPHIC DATA
94 patients with AMI were investigated among which 66
were male and 28 were female. The youngest patient was 33
years and the oldest was 94 years old. The median age was
67 years. 25% of patients were below 56 years old. (FIGURE
4-4)
IL GENERAL BIOCHEMICAL DATA
1). PLASMA CREATINEVE ON ADMISSION
The mean creatinine level was 111 ^mol/l in this
77 group of patient. It indicates that a high proportion of
patients have poor renalftinction. Ther e are 50% of
patients showing a creatinine level of great than 100
^irnol/l. (FIGURE 4-5)
2). PLASMA ALBUMIN ON ADMISSION
Most of the patient showed a low albumin on admission. More than 90% of patients had an albumin value less than 40 g/L while the median value was 33 g/L.
(FIGURE 4-6)
3). HAEMATOLOGICAL MARKERS OF NUTRITION
STATUS
� Haemoglobin
Almost 25% of patients were anemic (Hb < 12 g/L).
(FIGURE 4-7)
� Mean corpuscular volume (MCV)
78 Few patients showed a low MCV suggestive of
microcytic anemia (5%). Furthermore less than 5% of
patients featured macrocytic red blood cells, which may
indicate folate/B12 deficiency. (FIGURE 4-8)
� White blood cell (normal range:4-10xl0^/L)
Less than 5% of patients showed a low white cell
count.
4). TRACE ELEMENT STATUS IN AMI PATIENTS
�MAGNESIU. M (normal range: 0.67- 1.01 ^imol/L)
10% of patients were found to have
hypomagnesium(less than 0.67 ^mioLl^). The mean
magnesium was 0.77 ^imolyT., which was significantly
lower than the control group( the mean magnesium of
control group was 0.89 ^molA., standard deviation was
0.128 [imolfL). (FIGURE 4-9)
�ZIN. C (normal range: 9.8 - 18.4 ^imol/L)
79 A high proportion (more than 60%) of the AMI patient showed a zinc level lower than the lower reference limit of9.8 ^imol/L).
However, given the fact that plasma zinc decrease during early phase ofAML We classified patients with a zinc level lower than the lower 25 percentile value of 8
|imol/L as having a marked reduction of semm zinc.
Therefore 25% of patients were classified having an adverse level of semm zinc. (FIGURE 4-10)
When compared to control group (mean of which is
11.3 ^imol/L), AMI patients showed a significant low zinc level by t test (p<0.05).
③.COPPER (normal range: 10.7- 25.2 ^imoLO^)
A high copper level was found in only a small proportion of AMI patients. Only 5% of patients has value greater than 24.5 p.mol/L. When the mean copper value was compared with the control group, there was no
80 significant difference. (FIGURE 4-11)
�SELENIU. M
The 5th and 95^ percentile value of selenium in AMI
patients were 0.65 and 0.99 p.mol/L. However there was
no difference between AMI patients and the control
group. (FIGURE 4-12)
III. PROGNOSTIC IMPLICATION OF SERUM
LEVELS OF THE FOUR ELEMENTS
A set of potentially important biochemical factors were analyzed for their effect on patients mortality. As the variables were continuous and not all are normally distributed, they were defined into dichomatous variables by using selected cut-off values (Table 4-17)
The lower or upper reference levels were used for albumin, copper, magnesium, zinc. Plasma creatinine value
81 greater than 200 jLimoLT. which indicating a significantly impared glomerular filtration rate was taken as a high risk parameter. As the serum zinc and selenium have neither an appropriate reference interval during AMT nor a population reference intervals, the patients at the bottom 25^ percentile values were compared to the rest of the group.
By cross table analysis with modified chi square statistics, those patients with a high serum creatinine and low zinc concentration showed higher mortality. (Table 4-18)
The associations were furtherly analyzed with both univariate and multi-variate regression. (Table 4-19 & Table
4-20) By univariate analysis, patients age was found to be associated with mortality in addition to high Cr and low zinc groups. After exclusion, of confounding effects by multivariate logistic regression, high age, high creatinine, low
82 low zinc were remained significantly associated with higher
mortality and high copper/zinc ration which showed
marginal significance in univariate analysis (Table 4-19)
became significant.
4.2.3 DIGOXIN GROUP
I. MAGNESIUM
There are about 20% of patients on treatment of digoxin
showing hypomagnesemia. 10% of patients had serum
magnesium levels below 0.628 ^imol/L
As a group, the mean magnesium level is lower than the
control, which was 0.78 and 0.89 |imol/L respectively.
II. ZINC
There are 25% of patients had a serum zinc level below 8
^mol/L. And almost 50% of them were below the lower limit
of reference range (9.8 ^mol/L). Although the mean zinc level
was 10.6 p,mol/L which was lower than the control group, the
83 difference did not reach statistical significance.
84 5 DISCUSSION
5.1 AMI PATIENTS AND TRACE ELEMENT STATUS
L MAGNESIUM
Hypomagnesemia was found in only approximately 10%
of patients with MI at presentation to the hospital. And prior
use of diuretic agents may contribute to hypomagnesemia.
Hypomagnesemia was not a predictor of higher mortality.
(Table 4-22)
II. COPPER
Although previous investigation have demonstrated
elevated serum copper levels in patients with AMI, we found
no difference between the AMI and the control group.
III. ZINC
We identified low plasma zinc as an independent risk
factor for mortality after AMI. Age and poor renal function
were two well known factors affecting mortality which were
also documented in the present study.(Table 4-21)
85 A plasma zinc level lower than 8 ^imol/L was associated with an increase in the chance of dying from AMI.
IV. SELENIUM
There was no difference between AMI patients and the control group. The results do not support the hypothesis that selenium level changes during acute myocardial infarction.
And a low level of selenium during AMI was not associated with higher mortality.
V. RISK FACTORS ASSOCIATED WITH
MORTALITY AFTER AMI
Age and impaired renal function were well known to influence the post-AMI survival. In addition, low serum zinc and high copper/zinc ratio were also significant for mortality in this study.
There are a number of hypothesis which account for the
86 importance of these trace elements during AML Zinc has been described to concentrate in the myocardial tissue to participate in the process ofrepair and thus a lower serum zinc may be an indication of the infarct size.[Speich.M et al, 1988].
On the other hand, as zinc is largely bound to albumin, a low zinc may be part ofresult of extravasation of albumin during acute phrase reaction. Therefore albumin was not an independent risk factor in multivariate analysis suggesting that this mechanism can not explain all the variation of serum zinc. As high copper to zinc ratio, which can be taken as a marker of acute phase reaction, is an independent factor for mortality, the role of acute phase reaction on the long term outcome of patient after AMI is likely present.
87 The low zinc level could be the result of long term
nutritional deficiency. Patients with marginal zinc intake
may become overtly hypozincemic during acute stress like
AMI. As it is reported that zinc deficiency did lead to a
large infarction size and poor survival in experimental
animal. [Amaud J et al, 1991；
Therefore multiple factors may exit to account for the
link between low zinc and mortality. Further study is
required to clarify the interplay of this factors and to derive
a risk model for AMI patients.
5.2 MAGNESIUM AND ZINC STATUS IN PATIENTS
RECEIVING DIGOXIN TREATMENT
Electrolyte imbalance, mainly of potassium and calcium,
could precipitate digoxin toxicity even in patients treated
with conventional dose of the drug. However,
88 hypomagnesemia is also an important risk factor fordigoxin toxicity, though it is rarely measured in patients treated with digoxin.
In the group of patients studied, the overall magnesium level is lower than the control. It may suggest a generally poor food intake in these patients who were mostly suffered from heart failure and arrhythmia. Poor food intake in long term would potentially lead to hypomagnesemia and potentiate digoxin toxicity. It is alarming that 20% of the patients were hypomagnesemia, and almost half of them were severe (less than 0.6^imol/L). Therefore, determination of serum magnesium is indicated in long term management of patient on digoxin and appropriate supplement should be given when hypomagnesemia is confirmed.
It is interesting to find that one fourth of patients had a low zinc level (below 8 ^imoL^). It may indicate a long term ongoing acute phase reaction, but it may be unlikely for these to stable patients. A poor nutrition status may be a
89 possible explanation for the low serum zinc. Due to the nature of the study, samples were not adequate for further analysis of other nutrition markers, such as prealbumin for assess the protein nutritional status of these patients. A prevalence study for nutrition markers in these patients may yield important information on these patients with chronic illness.
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99 LIST OF TABLES
1. Table 1-1 RECOMMENDED DIETARY EMTAKES OF
MAGNESIUM, ZINC AND SELENIUM
2. Table 1-2 RECOMMENDED DIETARY INTAKES OF
COPPER
3. Table 1-3 REPORTED SELENIUM CONCENTRATION
IN SPECIMENS FROM HEALTHY ADULTS
4. TABLE 4-1 INTRA-ASSAY PRECISION OF ZWC
ANALYSIS
5. TABLE 4-2 BETWEEN-BATCH PRECISION OF ZINC
ANALYSIS
6. TABLE 4-3 ACCURACY OF ZINC ANALYSIS
7. TABLE 4-4 RECOVERY OF ZINC ANALYSIS (LARGE
VOLUME OF STANDARD ADDED)
8. TABLE 4-5 RECOVERY OF ZINC ANALYSIS (SMALL
VOLUME OF STANDARD ADDED)
9. TABLE 4-6 UNEARTTY & DETECTION LIMIT OF
ZmC ANALYSIS
100 18. TABLE 4-16 RESULTS OF CONTROL GROUP
19. TABLE 4-17 CLASSIFICATION OF ADVERSE
BIOCHEMICAL FACTORS
19.TABLE 4-18 RESULTS OF CHI-SQUARE ANALYSIS OF
THE RELATIONSHIP BETWEEN ^DIVIDUAL
ADVERSE FACTORS AND SURVIVAL AFTER AMI
20.TABLE 4-19 UNIVARIATE RELATION BETWEEN
VARIOUS DEMOGRAPHIC, CLMACAL， AND
BIOCHEMICAL VARIABLES AND MORTALITY AFTER
MI BY LOGISTIC REGRESSION (outcome as died)
21.TABLE 4-20 MULTIVARIATE RELATION BETWEEN
VARIOUS DEMOGRAPHIC，CLINACAL，AND
BIOCHEMICAL VARIABLES AND MORTALITY AFTER
MI BY LOGISTIC REGRESSION (outcome as died)
102 LIST OF FIGURE
1 FIGURE 1-1 EXCITATION AND DECAY PROCESSES
2 FIGURE 1-2 THE ATOMIC ABSORPTION PROCESS
3 FIGURE 1-3 BASIC AA SPECTROMETER
4 FIGURE 4-1 L_ARITY OF ZmC ANALYSIS
5 FIGURE 4-2 LD^ARITY OF COPPER ANALYSIS
6 FIGURE 4-3 LD^IEARITY OF SELENIUM ANALYSIS
7 FIGURE 4-4 AGE DISTRIBUTION OF AMI GROUP
8 FIGURE 4-5 SERUM CREATININE DISTRIBUTION OF
AMI GROUP
9 FIGURE 4-6 SERUM ALBUMD^^ DISTRIBUTION OF
AMIGROUP
10 FIGURE 4-7 SERUM HAEMOGLOBD^ DISTRIBUTION
OF AMI GROUP
11 FIGURE 4-8 SERUM MCV DISTRIBUTION OF AMI
GROUP
12 FIGURE 4-9 SERUM MAGNESIUM DISTRIBUTION OF
AMI GROUP
103 13 FIGURE 4-10 SERUM ZmC DISTRIBUTION OF AMI
GROUP
14 FIGURE 4-11 SERUM COPPER DISTRIBUTION OF
AMI GROUP
15 FIGURE 4-12 SERUM SELENIUM DISTRIBUTION
OF AMI GROUP
104 Table 1-1 RECOMMENDED DIETARY ENTAKES OF MAGNESIUM, ZD^C AND SELENIUM ‘
A^ Magnesium Zlm Selenium (years) (mg) (mg) (^ig) Infants 0.0-0.5 40 5 ‘ fO 0.5-2.0 60 5 15 Children 1-3 80 10 20 4-6 120 10 20 7-10 170 10 30 Males 11-14 270 15 40 15-18 400 15 50 19-22 350 15 70 23-50 350 15 70 51+ 350 15 70 Females 11-14 280 12 45 15-18 300 12 50 19-22 280 12 55 23-50 280 12 55 51+ 280 12 55 Pregnant 320 15 65 Lactating 355 ^ 7_5 Table 1-2 RECOMMENDED DIETARY STAKES OF COPPER
Age Copper (years) (mg)
Infants
0-0.5 0.4-0.6
0.5-1 0.6-0.7
Children & adolescents
1-3 0.7-1.0
4-6 1.0-1.5
7- 10 1.0-2.0
11+ 1.5-2.5 Adults 1.5-3.0 TABLE 4-1 IMTRA-ASSAY PRECISION OF ZJNC ANALYSIS
Low Median High (^mol/L) (^mol/L) (^mol/L) nJ0 21.41 32.36~~‘ 12.15 21.04 32.74 12.17 21.24 32.91 12.22 21.29 32.70 11.89 21.42 32.78 11.96 21.23 33.31 11.86 21.34 32.67 11.94 21.51 32.99 12.43 21.35 32.94 12.74 21.60 33.00 12.85 21.25 33.32 12.18 21.36 32.78 12.42 21.15 32.85 12.41 21.60 32.58 12.72 21.03 32.61 12.15 21.36 34.30 12.62 21.46 32.62 12.32 21.22 33.22 12.38 21.62 34.14 12.30 22.22 33^ TJ lo ^ ^ ~~ Mean 12.29 21.39 33.03 SD 0.279 0.252 0.508 CV 2.3% 1.2% 1.5% TABLE 4-3 ACCURACY OF ZmC ANALYSIS TEQAS* Target mean Target result Accuracy (^imol/L) (^imol/L) l40 fl7 8^ • 107.9% 441 11.00 12.05 109.50/0 442 22.93 24.39 106.4% 443 23.87 24.06 100.8% 444 11.60 12.38 106.7% 472 31.96 33.34 104.3% 474 14.66 15.13 103.2% 484 23.28 24.07 l03.4% 485 8.44 8.49 100.6% 486 32.82 33.76 i02.9%
* TEQAS: External quality control sample from UKEQAS trace elements scheme TABLE 4-4 RECOVERY OF ZDMC ANALYSIS (Large volume of standard added) Neat sample 6.12 ^mol/l Zn added 10.20 ^imol/L Zn added Zn conc Zn conc Recovery Zn conc Recovery (jumol/L) (^mol/TL) (^imolA.) ~5J~l 13.55 — 131.4% 13.59 79.2%" 7.27 16.15 145.1% 19.01 115.1% 5.57 13.75 133.7% 17.18 H3.8% 7.58 14.27 109.3% 18.29 105.0% 7.968 15.22 118.5% 19.01 108.3% TABLE 4- 6 LINEARITY AND DETECTION LIMIT OF ZWC ANALYSIS Expected concentration Results ~0 — 0.055 * ‘ 0.42 0.101 * 0.52 0.699 * 1.04 1.896 1.56 2.232 2.08 2.848 2.6 3.06 3.12 • 3.973 4.15 5.247 5.19 6.041 6.23 6.969 8.31 9.421 10.39 11.14 12.46 13.13 14.54 14.47 16.62 17.09 18.69 18.88 21.95 21.95
31.27 29.02
40.59 37.31 45.25 44.86 49.91 49.48 54.57 54.33 59.23 58.28 * RSD> 10% TABLE 4-7 WITHIN-BATCH PRECISION OF COPPER ANALYSIS
Low Median High (^iniol/L) (^imol/l) (|umol/L) ^ 19.40 3i:40 8.82 19.80 35.70 9.11 19.80 35.30 9.03 20.00 35.80 8.89 20.00 35.90 9.12 20.20 36.50 9.20 20.20 36.10 9.00 20.20 36.30 9.17 20.50 36.10 8.89 20.40 36.40 8.99 20.70 36.30 8.85 20.30 36.30 9.02 20.40 36.20 9.02 20.50 36.10 9.15 20.70 36.00 9.00 20.40 36.30 8.91 20.70 36.40 8.94 20.60 36.40 8.88 20.80 36.30 ^ 20.50 36.30 Tf W ^ ^ Mean 8.99 20.31 36.11 SD 0.108 0.353 0.325 CV 1.2% 1.7% 0.9% TABLE 4-8 BETWEEN-BATCH PRECISION OF COPPER ANALYSIS
Aqueous QC Serum QC (^imolyl.) (^mol/L) 1^ m 16.51 18.59 15.5 19.98 16.1 20.21 16.74 20.58 16.45 19.33 15.32 20.73 15.43 18.87 16.89 18.48 15.78 19.53 16.41 20.21 15.34 18.76 16.77 19.08 15.23 18.97
Tf H H Mean 16.13 19.51 SD 0.59 0.74 CV 3.6% 3.7% . TABLE 4-9 ACCURACY OF COPPER ANALYSIS TEQAS Target mean Target result Accuracy (^mol/L) (^mol/L) 1S0 r^ m 92.2% 441 30.62 29.7 97.0% 443 33.52 32.4 96.7% 444 27.60 25.8 93.5% 448 25.28 23.6 93.4% 473 30.38 28.7 94.5% 474 26.45 25.2 95.3% 484 23.99 22.2 92.5% 485 29.87 28.5 95.4% 486 22.72 21.3 93.8% TABLE 4-10 RECOVERY OF COPPER ANALYSIS (Large volume of standard added) Neat sample 6.29 ^mol/l Cu added 10.49 ^imol/L Cu added Cu conc Cu conc Recovery Cu conc ~~Recovery~ (|umol/L) (jLimol/L) (^unoLOL) T49 rr? 98.7% Kl "i02.1% 7.14 13.4 99.5% 17.8 101.2% 8.96 15.2 99.2% 19.9 104.3% 10.6 16.9 100.2% 21.4 103.0% 12.3 18.2 93.8% 23.0 102.0% TABLE 4- 11 LENEARITY AND DETECTION LIMIT OF COPPER ANALYSIS Expected concentration Results
0.00 — 0.05 * “ 0.43 0.05 * 0.54 0.21 1.09 1.02 1.63 1.63 * 2.17 2.39 * 2.71 2.97 3.26 3.68 4.34 4.73 5.43 5.61 6.51 6.79 8.68 9.04 10.85 11.10 13.02 13.40 15.19 15.50 17.36 17.40 19.53 20.20 17.70 17.70 25.50 25.50 29.40 30.00 33.30 34.10 37.20 38.00 41.10 42.20 45.00 46.30 * RSD> 10% TABLE 4-12 Within-batch PRECISION OF SELENIUM ASSAY:
Mean~~N ^5~~^"~Mean+2SD ‘ (p-mol/L)
High value: 1.145 22 0.07 1.3% 1.005〜1.285
Medium value: 0.513 21 0.02 3.3% 0.473〜0.553
Low value: 0.113 22 0.002 1.8% 0.109-0.117 TABLE 4-13 BETWEEN-BATCH PRECISION OF SELENIUM ANALYSIS Mean~~~N ^5~~CV Mean+2SD “ (p,mol/L)
High value:""TJU~~fs~~0.051 4.2 % 0.130-1.300 ‘
Medium value: 0.500 18 0.020 4.0% 0.471-0.525
Low value: 0.111 11 0.008 7.2% 0.097-0.120 seronorm: 0.267 11 0.009 3.4% 0.254〜0.283 TABLE 4-14 RECOVERY OF SELENIUM ASSAY
Sample standard Expected detected recovery (0.270 ^imol/l) ( 0.672 ^imol/l) value value
(1) 0.3 ml 0.1 ml 0.371 0.382 103%
(2) 0.3 ml 0.2 ml 0.431 0.422 98%
(3) 0.3 ml 0.3 ml 0.471 0.445 94%
(4) 0.3 ml 0.4 ml 0.500 0.479 96%
(5) 0.3 ml 0.5 ml 0.521 0.537 103% Table 4-15 LINEARITY AND DETECTION LIMIT OF SELENIUM ANALYSIS
Concentration Absorbance (H,mol/L) 0.009 -0.0020 0.012 0.0000 0.021 0.0029 0.039 0.0055 0.076 0.0107 0.082 0.0115 0.185 0.0261 0.233 0.0330 0.244 0.0345 0.385 0.0544 0.389 0.0550 0.486 0.0687 0.504 0.0711 0.890 0.1257 0.896 0.1265 1.120 0.1580 1.670 0.2360 2.270 0.3206 3.770 0.5322 4.430 0.6258 6.340 0.8954 7.790 1.1009 9.140 1.2910 10.050 1.4794 12.500 1.7600 15.900 2.2443 19.100 2.7035 23.700 3.3436 TABLE 4-16 RESULTS OF CONTROL GROUP
Element N(group size) Minimal Maximal Mean (SD) 10"】percentile 90'^ percentile value value Magnesium~"~40 0^^ YJl 0.9(0.13) 0.24 L^ (mmol/L) Zinc ^^ To 17.16 11.3(2.9) 7M TSM mol/L) Copper~~~^"^ ^ 1^ 18(3.47) 1^ 22.79 mol/L) Selenium (^i ~57 0^ L65 0.87(0.175) 0^ L^ mol/l) I TABLE 4-17 CLASSIFICATION OF ADVERSE BIOCHEMICAL FACTORS Adverse factors Define Remark
Low albumin Albumin < 30 g/L Lower reference limit
High copper Copper > 25g/L Upper reference limit
Low magnesium Magnesium < 0.67 [i Lower reference limit mol/L
Low selenium Selenium < 0.72 ^i Lower 25^^ percentile niol/l ofAMI
Low zinc Zinc < 8 ^mol/L Lower reference limit
Hcopper-zinc Cu-Zn > 2.2 Upper 25^ percentile ofAMI
Hcreatinine Creatinine > 200 ^ Poor renal function . molA^ TABLE 4-18 RESULT OF CHI-SQUARE ANALYSIS OF THE RELATIONSHIP BETWEEN INDIVIDUAL ADVERSE FACTORS AND SURVIVAL AFTER AMI Adverse factors Chi-square value Fisher's exact test
High Creatinine 11.2 *
High copper 2.9
Low magnesium 0.11
Low zinc 3.9 *
Low selenium 0.959
High Cu/Zn 1.99
* P<0.05 TABLE 4-20 Multivariate Relation Between Various Demographic, clinical, and Biochemical Variables and Mortality After MI ’
Factors . |P: ., Exp(B)
Age 0.0008 0.846
High creatinine 0.015 0.038
High Cu/Zn 0.048 12.45
Low zinc 0.0072 34.99 Table 4-21 SURVIVAL 一 LOW ZINC CROSSTABULATION
Low zinc Normal zinc Total "D^ ^ f3 n~~
Survival 14 56 70
Total 23 69 92~~" Table 4-22 SURVIVAL-LOW MAGNESIUM CROSS TABULATION
Low Mg Normal Mg Total Dead Tl 3 ^^
Survival 63 7 70
Total 84 10 94 EXCITATION —•— (1) Er>«rgy 十 • ^ Ground ^^“^ SUte ST.t« Alom A，°^
DECAY . � ztz — Z：^ + ^ ^crto^ Ground Light ^»'® Sute Er>ergy Atom Alom
Figure 1-1 Excitation and decay processes
(adapted from Richard D, 1993)
�_ =^ —亡 ，t Ground Excrt^ ^f^f5y SUta suta Atom Atom
Figure 1-2 The atomic absorption process
(adapted from Richard D, 1993) I Ligh.Soorc | S.r^«C.« | Sp«crtk:L_M^,u™a^n<
1 £?1~KQrff^~f)H>^_ i I C.hoppGf I / \ I Uonocrtrcmalof &^tor P^
Figure 1 -3 Basic AA spectrometer
(adapted from Richard D, 1993)
\ Figure 4-1
Plasma Zinc Linearity Study
50 ； i ； ； ^ i ! //// ；- z// : -t z _ „ ；； ,/
i 30 ；- :/^ -
I«««^, / )/ i ： Z
^ 20- / _ OJ ‘ y/ • ^ i z s / i 10 - / _
:/ :. [z i 〇 i^ i~:~~^~~^ ！~~：~_：_一_： , . . i ‘ 0 10 20 30 dO 50 Expeccea (umoLyL) �
�• 131 Figure 4-2
Plasma Copper Linearity Study
50^ 7,
1 ./1 r Z -i Z I L :f ！ i 4^ \ !- ^ i 。 L Z I ^ I y/ i 0 30 - :z ! 三 ：_ -j;;^ ‘ -:~ ,z : ~： I 1A ‘ 1 - Z 二。 ‘ y/ 03 90 — 0^ ^ /
^ - / � Z ： L _j< , i 7 i 10^ / ^1 / I n "f^ - . ‘ • ‘ • • •
0 ^ i i ； ‘ I 丨‘ i. 0 10 20 . 30 dO 50 • • Expected (umol/L) s
• • • . 、'^ � Figure 4-3 Linearity of selenium analysis
^QDDDr ( 35TOF 4 •岁 3CDDD^ • •• 23TOr 令• • 2猶 • g i y < i,y Iaffir �• 身 — 參.梦 Q5DD^ 1^ y QCDDC^^ 1 QCn> oCro DGCD SOD 20D SCQ) 3)OD
-*} 3-tD 1~-~~_.—趣—.一"~~~-.»—墨.• • - I 乂 concentration
133 Histogram 20 ？
j f^T^^ ^r^^g:^S¾ ^ j?»fe^^^^S^ ^a^£^^«^3 ^^^^^^ s^ ^Sx _ ^^^^^�JJ :(5^^J¾ :Q- :^E3/ -•• ^|^^j!^rii^^^^B ~E"•^•^^^/ ••.-_ • ,. -*J^1:J^^"7S^^^[^¾¾!'>',1^7^^^^¾¾^^¾ j-,>r_??^-^'^^ . ,y'"^ ^^^^^^^^^^^^¾ 囊 ^^^^^^^纏 ^¾ CLSSjjj|jg^j^^j^|i^F^^P t^M^ /�d ^;;^¾^¾¾¾¾^¾^^¾¾¾¾??^ . 11 ^^M'-x :=:， LL. 0 ’yr_if，-〒?r^^:pi=flg^fejtffi,〈^'i_i |丨|_|_‘闲 N = 94.00 35.0 45.0 55.0 55.0 . 75.0 . 35.0 . 35.0 40.0 50.0 30.0 70.0 80.0 90.0 . FIGURE 4-4 AGE DISTRIBUTION OF Ai\tI GROUP Histogram 40
‘ I ； ；
‘ M 30 -‘ _ 3??ttnilr^SiSsaj CTTji*^ff^t>aPp|T|'B*wa 」、1 抽:; 化 20- 圓 5;¾¾&¾ a&tu&^^4jff3SeS^^faSS&fW^l ^SSs^3L 、 ws^k^M^¾! ^ ,s^^ 岂 ''/" a^B \ iStd. Dev = 60.5l Z \ W|^WphB \ |Mean = 111.3 ^ 0 I: I ^^^^^^^PmnB ^^w i N = 91 00 嫩聯滅微》，^^。.
FIGURE 4-5 SERUM CREATININE DISTRIBUTION OF
AMI GROUP Histogram
‘ . 16- ： • I丨 ， 14’ m m ,: I I ^ 2 - 碧 靈 I圓-
1�� I I I - |ti '• I U\ ^ “ al 圓 B| | i 2i m 1 iiM#ffl :Std.Dev = ^1C g" ^^y!j'y_l|Wip|i|jijJ|||H|H^ ! '^ean = 33.4 U： 0 ^"^^Bj[l|fflj||S:|'Jl'iB^J^WIpBI^"^^"^ 謂 N = 97 00 %%%%^；%\%\\%% -
FIGURE 4-6 SERUM ALBUTvD[N DISTRIBUTION OF
AMI GROUP Histogram
r^ i r^^^^rtT^ mtnm I P^f?^ ^¾ ！ ^jrj'j^L?^^^¾ ^ c*v%圏j i^iyi7Jffl ^M I ^ij^jgjgl^l^ 10r 'T"*"""*Tpii^..
^^¾^?^);)¾^<¾¾¾¾¾¾¾¾^�‘ >, I ^^^^^¾¾¾ \ C I / a jJ^^M , cu ^jj^^^^^llli^iii',',ll'^ffVyf^l! jji^Sj^HuRil� Std. Dev = 173 g- / MMMto^llllliff''!!lfl"i^ ‘ Mean = 13.7 1 0^ _^^^ifjPilibiil^iifB^pi:N = 77.oo 7.0 9.0 11.0 13.0 15.0 17.0 8.0 10.0 12.0 14.0 16.0 18.0
FIGURE 4-7 SERUM HAEMOGLOBIN DISTRIBUTION ‘
OF AMI GROUP Histogram
20^ ； , I
—«^Bm1 10」 _HBwi ^^^^^S^^^BmBa > ！ Ji\ _ /^MB^K^ |sW.Dev = 5.S8 g- / ,^MMpMlXi ^^ean = 39.4 圣 0 ^S_J_^^^^!;t__liifc N = 76.00 62.5 67.5 72.5 77.5 82.5 87.5 92.5 97.5 65.0 70.0 75.0 80.0 35.0 90.0 95.0 100.0
FIGURE 4-8 SERUM MCV DISTRIBUTION OF AMI
GROUP Histogram 40- ； I I , I ； j • i 尋 g^*^ ^¾^^ ^f7g|•^¾¾?憩 g^^ | 20 -' ^¾ ^j^ ^¾¾¾ y^j*T^!w.'rtsg彻卞a rJanal押 ttfiSai i^jjS|j*8|^^ ^^S|8^mmm^ftgCiai\ i 圓 ^|^i^ o> 飞1。 n“； I 圔^^彌味！ »rCTI^溯v 广 i l-i^tf^tmMttQtS; ^^¾¾ \ 5 1 gss|j^!ji^g5|g \ Std. Dev = 11 o7j ！ ^^^^iifliHiriT'jWWfBw^a^aB/ ‘/''ili'i^MwiBTOHM Mean = 77 i： 0 I«m I Jplf^lli "f|'|'|i'|||l|||^l^g N = 94 QQ 19 .31 .44 .55 . .69 ‘ .81 ‘ .94 .25 .38 .50 .53 ,75 .88
FIGURE 4-9 SERUM MAGNESIUM DISTRIBUTION OF '
AMI GROUP . 12- ! ！ i1 I
10- ！ ：：I fL 4- i^m L ； jj|{Uj|j^|||^mi^^^^^^ 2丨，iiliHimi\^ Std Dev = 2.61 |M mH^BBBgj^B^B|^B^m l^^^m _Mean = 9.96 0 iajwm_ii___Lmui N=92.00 “ �����wwww
FIGURE 4-10 SERUM Zi:NC DISTRIBUTION OF AMI
GROUP
� Histogram 30-—— ^ i, ：( [j^jg I H i 1¾^¾ ！ 20- •^3 I\
！ M\ / Wr^n9^f^ ^m ^M^Mgj^^- 1。-' ^^H >N H^9^^^HSSI^SB^^^BEs c I ^B^H^B .� ? /9^^^9BE_2x :Std, Dev = 3.93 ^ .[_^ 4^^mBnffl!/g 1Mean=i6.7 ^ ° ^^^ ^^^^^^^lllHil'ii' IOiil'|'[^'^"-^eamH' N = 94.00 S-° 则 14.0 18.0 • 22.0 ‘ 27^^^
8.0 12.0 16.0 20.0 24.0 28.0 32.0
FIGURE 4-11 SERUM COPPER DISTRIBUTION OF ‘ AMI GROUP Histogram
12- I 室 10二 g^^K^ • i ？^^ 園 -.>^>y.-f-'<'l!J| ^gpJ “ •動\ 3 _ ^^B 7~"口 1^ ^H ^^^B\
-•!^:广：二厂.， “—^^^^^^^I^^^^Jjj^j^ \� ^?^VV' Z^^'l•^ jMjtffWHM ^B 4- ^^ ""^^^^^^^^^¾^ _ 一 ^ 1 ^g^;^^m^. I § 2. . B-n^^^MjiM >^|-ji ,Std.Dev=.1G
^ 丨 乂,..._^^P^^^^""=^=^^,___M ^^g Mean = 789 i 1 —r，「_ I il|_|||||| , fCT W^W^W^W^W^V-/% FIGURE 4-12 SERUM SELENIUM DISTRIBUTION OF AA4I GROUP ‘ .“、:磁绝:《 EbD5DiEDD 11_隱^^^^ ssLJBjqtn >|Hn3