Estimation of Lipoprotein Ratios and Fractions

Used in MPhil thesis calculations and analysis [Population Variation for Risk Factors in Ischaemic Heart Disease, 1992].

Estimation of LDL-cholesterol (LDLc) using the Friedwald Formula. [Friedwald, W. T. et al. Estimation of the concentration of low density lipoprotein cholesterol without use of the preparative centrifuge. ClinChem. (1972), 18 (499-502].

Original Friedwald Formula (FF) = LDLc, mmol/L = TC – HDLc – (TG x 0.45).  LDL-c, mg/dL = TC – HDLc – (TG x 0.20).

Estimation of total cholesterol (TC) = TC = VLDLc + LDLc + HDLc.

Lipid Classification – total lipids cholesterol (HDL + LDL) + triglycerides  (VLDL).  Measurements of these cholesterol fractions can be obtained by knowing two of them from the following formula:

LDL = TC – TG/5 – HDL (in mmols).  [See : Graham, M.  (1988). The Effects of Cholesterol.  The Physician].

Also – LDLc = TC – HDLc – TG/2.19 (mmols).  [See:  Day, R. et al. (1989).  Treatment for hypertriglyceridaemia – state of the art.  Card in Pract. Feb].

Modification of the original FF.  See: DeLong D. M. et al.  (1986). A comparison of methods for the estimation of plasma low- and very low-density lipoprotein cholesterol.  JAMA 256 (11). Nov.

The DeLong modification of FF. LDLc, mmol = TC – HDLc – (TG x 0.37).   LDLc, mg/dL = TC – HDLc – (TG x 0.16).

They concluded that LDLc was better estimated using the following formula:

LDLc = TC – (LDLc + 0.16 x TG). mmols.   Or:  LDLc = TC – (HDLc + 0.37 x TG).  mg/dL.

This proposed formula is more precise for plasmas or sera with a TG concentration within the normal range.

Thus the original FF = plasma TG in mgms/L is determined and an estimate of VLDLc in mgms/L is made by: dividing TG by 5 (i.e E-VLDLc = 0.20 TG), (0.46 in mmols). DeLong called estimated VLDLc – E-VLDLc.

Therefore – estimation of E-VLDLc = E-LDLc = TC – (HDLc + E-VLDLc) = TC – (HDLc + 0.20 X TG)

Modification of the original FF using an intercept: the linear regression coefficient for VLDLc on TG and the intercept.

See: Rao, A. et al.  (1988).  Calculation of low-density lipoprotein cholesterol with use of triglyceride/cholesterol ratios in lipoproteins compared with other calculation methods.  Clinical Chemistry.  34 (12).

Therefore – LDLc mmol/L = TC – HDLc – (TG x 0.342) = 0.118.  Also – LDLc mg’dl = TC – HDLc – (TG x 0.148) + 4.6.

Using an FF that varies with TG: FF was found to correlate significantly with TG. (FF = 0.47 – (0.022 x TG).

Therefore – LDL mmol = TC – HDLc – TG = [0.47 – (0.022 x TG)].  Thus – LDL mg’dl = TC – HDLc – TG = [0.022 – 0.00011 x TG)].

Rao, A. et al (1988).

 

 

 

 

 

 

 

To be continued

Sociology of Preventive Medicine – research proposal.

Proposal and Perspective

Sigerist claimed that medicine was basically not only a social science but also an occupation – whose practice was inextricably woven into society, thus pointing out their inter­dependence. This interdependence can be seen in one branch of medicine that has built in socialemphasis – that of public health and, preventive medicine.

Freidson (in The Profession of Medicine) stressed the point that the jurisdiction of the medical profession over ‘illness’ labels can be categorised in a number of ways. One category of some importance to preventive medicine is the “expansion of what in life is deemed relevant to the good practice of medicine”. Furthermore, this implies that medicines commitment to specific aetiological models of disease has changed to one of multi-causal orientation. The greater acceptance of concepts of comprehensive medicine has therefore expanded that knowledge relevant to the prevention of disease. In prevention the expression ‘extension into life’ implies the very idea that primary prevention means getting there before disease starts. It is at this point that forms of social inter­vention and control emerge for the purpose of disease control

Freidson has developed the perspective (The Profession of Medicine) that the sociology of medicine has the purpose to anal­yse illness as a form of social deviance, as well as the scientific study of behaviour surrounding illness. In this respect therefore Zola’s view (Medicine as an institution of social control) could be incorporated into the research framework. More specifically as a topic entitled preventive medicine as a form of social intervention and control with reference to….”

For example, areas amenable to study by such a frame­work could include maternity services, peri-natal medicine, ischaemic heart disease and primary care, non-accidental injury (baby battering syndrome), and cancer, as well as aspects of health education. Using the continuous process of literature reference and review (as outlined by Conway and McKelvey in ‘The Role of the Relevant Literature: A Continuous Process, Journal of Educational Research, 63, (9), it would be possible to expand study of peri-natal medicine to include related topics (amniocentesis, clinical abortion, genetic counselling, and attitudes to screening etc).

A sociological perspective of preventive medicine as a form of social control would necessarily involve the determination of professional outlooks (attitudes, role conceptions, ethical consider­ations) as well as determine the attitudes, expectations, beliefs of patients and prospective patients. The embryonic model outlined above requires further development and clarification. To this purpose I am in the process of reading the following books in order to construct a firmer proposal, especially in respect of preventive medicine.

References and Sources Consulted

Medical Sociology: A Selective View. (D. Mechanic).

H.Sigerist on the Sociology of Medicine. (ed M. Roemer).

Politics, Medicine and Social Science. D. Mechanic).

Sociology and Medicine. (Susser & Watson).

Patients, Practitioners and Medical Care (D. Robinson).

The Process of Becoming Ill. (D.Robinson).

Social Science and Social Pathology (B. Wootton).

 

Originally accepted , after interview, by the University of Aston (for 1975), with offer of accommodation, plus cooperation of local peri-natal and post-natal services, but not pursued because PhD research studentship not available after financial cut-backs..

The Apoproteins As Genetic Markers In The Early Diagnosis Of Hypertriglyceridaemia

Introduction

This essay has been written as a contribution to the discussion concerning the role played by plasma lipids in the aetiology of coronary heart disease. Evidence is now regarded as conclusive that there is a definite correlation between raised plasma lipid levels and the onset of ischaemic heart disease, and that appropriate therapeutic intervention lowers this risk *.

*The Lipid Research Clinics Coronary Primary Prevention Trial Results. Reduction in Incidence of Coronary Heart Disease. JAMA. 251:351-364. 1984.

The underlying theme of this essay is that the apoproteins may serve as better, even more useful, indicators of the existence of familial hyperlipidaemias (Avogaro et al, 1979). Especially because the determination of plasma apolipoproteins may enable earlier diagnosis to be made – hopefully amongst younger age groups than is at present possible. In this context special consideration will be paid to Type IV familial hypertriglyceridaemia which at present defies pre-adulthood detection by current methods.

Furthermore, consideration will be given to present knowledge of the inheri­tance of Type, IV hyperlipoproteinaemia and current research into the determination of associated apoproteins as genetic markers for the condition. Finally there will be a proposal for further avenues of research that are aimed at determining apo­protein levels that may enable the detection of familial hypertriglyceridaemia in younger age groups. The specific theory underpinning this essay is that, even though plasma lipid levels indicating Type IV are difficult to demonstrate amongst young persons, the apoproteins related to the condition may prove to be a more reliable diagnostic tool and possibly demonstrable in children and adolescents.

The apolipoproteins – a review

Many proteins require, for their biological activity, tightly bound and specific non-polypeptide units. Such units are termed prosthetic groups. Thus a “…protein without its characteristic prosthetic group is termed an apoprotein.” (Stryer, L. 1974). All lipoproteins contain proteins and the apolipoproteins represent the lipid free components of plasma lipoproteins. Chylomicrons (fat globules) were the first lipoproteins discovered. Apolipoproteins are obtained by treatment of intact lipoproteins with detergents, organic solvents, and chaotropic agents. It is known that the proteins consistently isolated with the chylomicrons are specific apoproteins. Furthermore, all lipoproteins that are isolated from liver and intestinal cells are seen to consistently contain one or more apoproteins. Lipoprotein densities “…are inversely related to their content of lipids relative to proteins, i.e,,thegreater the lipid content, the less dense their particles.” (Schonfeld, G. 1983, 91). There occurs in the circulation an extensive exchange and net transfer of apolipoproteins and this “…contributes considerably to the apolipoprotein content of serum VLDL.” (Herbert, F. N. et al, 1983, 592). It is also known that some apoproteins are confined to HDL, some are confined to LDL and VLDL, and that some are shared by all forms of lipoproteins. And moreover “…the apoprotein compo­sitions of intestinal lymph and plasma chylomicrons are different.” (Schon­feld, G. 1983, 91).

With  regard to the nomenclature of the apoproteins they were originally named by their individual discoverers. In recent years an ABC nomenclature was proposed (Alaupovic, P. 1971) and ^,this classification has since been adopted by most researchers in the field. The nomenclature is based upon the hypothesis that plasma lipids are transported by different families of lipid-protein complexes (Alaupovic, P. 1972). The apoproteins that are of importance in lipid transport are those in the series apoA to apoB. For example, the lipoprotein family A consists of complexes of lipids and the apoproteins A. Lipoproteins isolated from plasma by ultra-centrifugation, are, according to this concept, either complexes of or single lipoprotein families. For example, virtually all of the lipoprotein B family are associated with LDL, and VLDL comprises a complex of B, C and E. The Roman letter and numeral designation was adopted by Alaupovic and then applied to all isolated single lipoprotein families. But, as has been noted above, the nomenclature is not universally accepted yet. For example, there is no apoA-I;II because other researchers named it apoD

The apolipoproteins have been studied according to their functions, origins, distribution, concentrations, molecular weights and polymorphisms. The primary structures of five of the apolipoproteins have been determined and these are A-II, C-I, C-II, and C-III, (Jackson, R. L. et al, 1977). Moreover, it has been shown that these proteins contain about the same proportion of non-polar and polar amino-acids as do other soluble proteins. This gave rise to the hypothesis that “…there are specific and specialised lipid-binding regions within these molecules.” (Herbert, P. N. et al, 1983, 593) because sequences of apolipoproteins do not contain long stretches of hydrophobic amino-acids, (Segrest, J. P. et al, 1974). These lipid-binding regions are described as amphipathic helices and have been shown to exist in the five apolipoproteins of which the primary structure is known (Sparrow, J. T. et al, 1975). These amphipathic helices are thought to enable apolipoproteins to form stable structures with polar phospho­lipids. It is thus probable that most apoproteins are bound to phospho­lipids and even other apoproteins “…on or near the surface…” (Schonfeld, G. 1983 93) thus approximating a monolayer on the plasma lipoproteins. Thus the “…outer layer or surface regions of lipoprotein particles are formed by hydrophilic molecules, the phospholipids and apoproteins, while the inner or core regions contain the hydrophobic, cholesterol esters, and triglycerides.” (Schonfeld, G. 1983, 93).

The apoproteins display a₄number of important metabolic functions. These functions are related to the specific domains found on the three-dimensional structures of individual proteins. All apoproteins bind to lipids. This is because they share the structural feature known as the amphipathic helix domain in common. The hydrophobic face of this helix is thought to interact with the hydrophobic and fatty acid portion of the phospholipids – whereas the helix’s hydrophilic face interacts with the phospholipids polar region. Researches have shown that the association of lipids and apoproteins is regulated by the law of mass action. This is because apoproteins may dissociate themselves from one lipoprotein and move to another, (Brewer, H. B. jr, 1981). Alterations in lipid-protein interactions appear to be routine during lipoprotein metabolism. Virtually all apoproteins (excepting apoB) are able to alter their plasma lipoprotein associations. In the case of apoB – this appears to remain associated with the same lipoprotein^–particles throuighout^– its^– metabolic life. We can outline the essential structure and functions of the major apoproteins.

Apolipoprotein A-I is a major protein component of primate HDL. The amino-acid sequences of the apoA’s are known and none of which are chemically related. Each possesses a different function (which may suggest a four gene complex) and apoA-I and apoA-IV are single proteins. ApoA-I consists of a single chain of between 243 and 245 ammino-acid residues (excluding cystine, cysteine, leucine and carbohydrate). Mutant forms of apoA-I have been recognised as have several isoforms (A-I, A-I₂ ). ApoA-I is a potent activator of Lecithin Cholesterol Acyl Transferase (LCAT) which is “…a plasma enzyme catalyzing the conversion of cholesterol and phosphatidylcholine to chclesteryl esters and lysophosphatidylcholine.” (Herbert, P. N. et al, 1983, 594). It is the specific lipid-binding domains of apoA-I that activates LCAT – this activity is associated with the property of lipid-binding.

ApoA-I is synthesised in the intestines and liver and has a molecular weight of 28,300. It has a plasma concentration of 90 to 130 mg/dl and more than 90% of plasma apoA-I is associated with HDL. Less than 1% is associated with VLDL and LDL. ApoA-I constitutes no more than 10% of the lipoprotein free fraction of plasma (Cheung, M. C. et al, 1977), and comprises a major (5% or more) component of chylomicrons and HDL, a minor portion of VLDL and a trace of LDL.

Apolipoprotein A-II is a major constituent of human HDL constituting about one third of the total protein and 15% of the HDL mass. ApoA-II has a molecular weight of 17,000 with a plasma concentration of 30 to 50 mg/dl. It is synthesised in the liver and intestines but its function is unknown. ApoA-II has a similar component distribution to ApoA-I in HDL and VLDL etc. This apoprotein exists as a dimer of two identical chains that comprise 77 amino-acid residues each but, however, the primary structure is unknown (Brewer, H. B. jr et al, 1972). Both forms of apoA-II (monomeric and dimeric) are capable of reassembling with phospholipid and specific lipid-binding segments have been identified (Mao, S. J. T. et al, 1970). In so far as lipid transport is concerned the specific role of apoA-II has not been determined, but it is established that the bulk of apoA-II is found in HDL but less than 55, in other lipoprotein classes

Apolipoprotein A-IV is found predominantly in the lipoprotein free fraction of plasma and lymph. It is synthesised in the liver and intestine and has a molecular weight of 46,000 (Beisieg, U. et al, 1979). ApoA-IV is a major chylomicron constituent (5% or more), appears as a trace in VLDL, as a minor component of HDL but is absent from LDL.

Apolipoprotein B is a constituent of chylomicrons (major), of LDL (major), and VLDL (major) and only a minor component of HDL. ApoB comprises more than 90% of protein LDL but little is known about its structure. There is evidence for heterogeneity for human apoB. It has been shown that apoB exists as a series of proteins in plasma LDL with the following types: apoB 100 (molecular weight 549,000); apoB-74 (molecular weight 407,000); and apoB-26 (molecular weight 126,000), (Kane, J. P. et al, 1980). ApoB-100 is the predominant species from which both apoB-74 and apoB-76 are derived, and this is indicated by size and amino-acid composition. It also appearsthat a second distinct type of apoB comprises a major constituent of the chylomicrons and which is not found in LDL and has an m/wt of 265,000, so “Immunological differences between the large and small varieties of apoB probably…exist…together with evidence for structural and chemical differences; suggest that they are products of different genes” (Herbert, P. N. et al, 1983).

The amino-acid sequence of apoB is unknown but it is thought that it may consist of four proteins (Kane, J. P. et al, 1980). Further to this apoB plays a role in the synthesis of both chylomicrons and VLDL – appearing to be critical to the receptor-mediated uptake of LDL. In addition there is also evidence for a role of apoB in LDL clearance as well as formation (Shepherd,  J.  et al, 1979., and Shepherd, J.et al, 1980). More than 907 of plasma apoB in normal subjects and in hypercholesterolaemic patients is found in LDL. ApoB in VLDL and chylomicrons constitutes between 20 and 507 of the total in moderate to severe hypertriglyceridaemia (Schonfeld, G. et al, 1974;  Albers, J. J. et  al, 1975).

Apolipoproteins C-I, C-II, and C-III are “…distinctive proteins with distinctive functions.” (Herbert, P. N. et al, 1983) of which the amino-acid sequences are known. ApoC-I is a single protein with a molecular weight of 6,500 to 7,000 (Shulman, R. S. et al, 1975). ApoC-I constitutes 107 of the protein in VLDL and 2% in HDL. Synthesised in the liver it can activate LCAT and bind phospholipid. It is a single chain protein of 57 amino-acid residues (Shulman, R. S. et al, 1975: Jackson, R. L. et al, 1974). ApoC-I is a major component of chylomicrons and VLDL, a minor constituent of HDL and occurs as a trace in LDL.

Apolipoprotein C-II (apoC-II) is synthesised in the liver and functions as an activator of lipoprotein lipase. It constitutes 10% of VLDL protein, between 1 and 2% of HDL-2 and less than 1% of HDL-3 (Kashyap, M. L. et al, 1977). ApoC-II is a single chain protein of 78 Or 79 amino-acid residues whose sequence is now known (Shulman, R. S. et al, 1975: Jackson, R. L. et al, 1974; Jackson, R. L. et al, 1974). As mentioned above apoC-II is a potent activator of lipoprotein lipase “…the enzyme catalyzing the hydrolysis of triglyceride in chylomicrons and VLDL.” (Herbert, P. N. et al, 1983, 595). The importance physiologically of lipase activation has been documented and where it was shown to-be (in one case of severe familial hypertriglyceridaemia) secondary to an absolute deficiency of apoC-II (Beckenridge, W. K. et al, 1978). This has to be considered against the normal situation where “…the quantity of apoC-II in plasma considerably exceeds that required for lipoprotein lipase activation.” (Herbert, P. N. et al, 1983, 596). ApoC-II has also been shown to occur in two forms – apoC-II-1 and apoC-II-2 (Havel, R. J. et al, 1979). It has a molecular weight of 8,800 to 9,000 (Jackson, R. L. et al, 1977a).

Apoprotein C-III. (ApoC-IIl) has a molecular weight of about 9,000 (Brewer, H. B. jr et al, 1974). It is the most abundant of the C proteins and constitutes about 50% of VLDL protein (Catapano, A. L. 1980) but also 2g of HDL. It is a single chain of 79 amino-acid residues of a known sequence (Brewer, H. B. jr. et al, 1974; Shulman, R. S. et al, 1974). ApoC-III occurs in three polymorphic or isoelectric forms – apoC-III-2, apoC-III-1, and apoC-III-0. They are classified according to the number of sialic acid residues at the end of the carbohydrate chain. ApoC-III-0 has no sialic acid content, apoC-III-1 has one molecule of sialic acid and apoC-III-2 has two molecules. (Vaith, P. et al, 1978). About 25% of apoC-III in normal plasma is associated with VLDL whereas 60% is in the HDL. However, more than 50% of the total apoC-III may be in the VLDL in hypertriglyceridaemic serum (Curry, M. D. et al, 1980).

Apolipoprotein D (apoD or apoAIII) is a protein of a molecular weight of approximately 32,000 and constitutes less than 5g of HDL apoprotein (Kostner, G. M. 1974). ApoA-III is Kostner’s designation but apoD derives from another group of researchers (McConathy, W. J. et al, 1973). ApoD is a minor apoprotein of HDL but its componency of chylomicrons is unknown. In VLDL it is a minor constituent if present at all and occurs as a trace in LDL.

Apolipoprotein E (apoE) comprises 10 to 20% of VLDL protein and has been detected immunochemically in all lipoprotein classes. It usually occurs as a single chain with a molecular weight of 35,000 to 39,000 (Rall, S. C. jr et al, 1982). Extensive heterogeneity has been reported. Three major forms, or isoforms, of apoE (E-2, E-3, and E-4) are thought to be products of three alleles at a single locus (Zannis, V. I. et al, 1981). ApoE heterogeneity has three sources. One source is genetic due to substitutions in its primary amino-acid sequence and the other is due to varying degrees of sialylation (Zannis, V. I. et al, 1981). It is thought that the recognition of apoE-3 and apoE-4 by hepatocyte receptors may provide a link in the normal conversion of VLDL remnants to LDL (Havel RJ et al, 1980). Furthermore, serum levels of apoE appear to correlate highly with triglyceride concentrations (Blum, C. B. et al, 1980). ApoE is synthesised in the liver and may be a receptor-mediated lipoprotein remnant of catabolism. The apoE receptor on hepatocytes is known to bind chylomicron remnants and serves as a recognition marker for the uptake of lipoproteins by several cell types.

Apolipoprotein metabolism.

In addition to the apoproteins there are certain enzymes and cellular receptors that play a role in lipoprotein metabolism. For example, beta VLDL and chemically altered lipoproteins are found on macrophages (Mahley, R. W. et al, 1980; Goldstein, J. L. et al, 1979). All receptors mediate the internalization of the lipoproteins they bind.

Dietary triglycerides and phospholipids undergo partial hydrolysation in the lumen of the gut and are absorbed together with cholesterol by the enterocytes. It is within the enterocytes that these lipids are re-esterified. These re-esterified lipids are assembled with specific apoproteins (apoB, apoA-I and apoA-IV) to form chylomicron particles. Intestinal lymph, which contains chylomicrons; is collected and enters the venous system via the thoracic lymphatic duct (Schonfeld, G. 1983). The chylomicron component of the lymph passes to the venous plasma where it undergoes a series of changes. Chylomicrons in the plasma acquire apoC proteins and apoE proteins by transfer from the circulating plasma HDL (Havel, R. J. et al, 1973). The triglycerides and phospholipids are also hydrolyzed by LPL (lipoprotein lipase) which is located on the endothelial cell surfaces of the arteries and capillary beds (Blanchette Mackie, E. J. et al, 1973; Higgins, J. M. et al, 1975). From this it can be deduced that an excess of circulating lipids will lead to excessive binding to the arterial and capillary walls.

Lipoprotein lipase interacts with apoC-II on the surfaces of the chylo­microns. This catalytic activity results in lipolytic products that include free fatty acids, lysophospholipids, and glycerol. These products are taken up by the tissues and thus form sources of energy and for membrane synthesis. Thus the “…net result of the intravascular catalysis and the movement of lipids and apoproteins is the conversion of chylomicrons to chylomicron remnants.” (Schonfeld, G. 1983). Smaller than the chylomicrons, these remnants are enriched with cholesterol and apoproteins. Important apoproteins in this respect are particularly apo’s B and E.

Inheritance, hypertriglyceridaemia and the apoproteins.

Hypertriglyceridaemia in its familial form demonstrates a “….wide variety of as yet unidentified biochemical defects, each of which produces a similar phenotype.” (Fredrickson, D. S. et al, 1978). It has not so far been possible to determine what proportions of familial (type IV) hypertriglyceridaemia are polygenic or monogenic, although the trait appears to act as a Mendelian dominant in some families (Murphy, E. A. et al, 1977). It does seem probable that the inheritance of type IV is polygenic. A developing trend of research is progressing along the lines of searching for genetic markers, especially those associated with the apoproteins.

When considering the apoproteins and type IV it is seen that there are many shared metabolic and physical properties between VLDL and chylomiccons. They serve thus as a transport mechanism for triglyceride. It is not known with any certainty, however, whether intestinal VLDL are more closely related to chylomicrons or to the hepatic VLDL. It has been said that “…the apoprotein composition suggests that chylomicrons and intestinal VLDL repre­sent a class of lipoproteins that should be distinguished from hepatic VLDL.” (Simons, L. A. et al, 1980). With regard to type IV and the search for genetic markers we can state that VLDL contains the major apoproteins apoB, apoC-I, apoC-II, apoC-III and apoE. VLDL only contains traces or minor constituents of apo’s  A-I and it is known that plasma VLDL contains approximately 8  to 10%  protein, _ithe principal ones being apo’s B, C  and E (Kane, J. P. et al, 1975).

It is the hypothesis of this essay that the techniques applied to the determination of genetic factors – especially using the apoproteins as genetic markers – could be applied more widely to hypertriglyceridaemia. A recent trial and study (Sveger, T. et al, 1983) suggested that the analyses of apolipoproteins, especially apoB, are of value to trace adolescents at risk for future coronary heart disease. In this study the subjects were tested for apoA-I, apoB and HDL. The conclusion was that it is “…well established that elevated total-C and/or elevated apoB as well as low HDL-C and/or apoA-I are risk factors for CHD (Sveger, T. et al, 1983 ), see also (Avogaro, P. et al, 1978: Whayne, T. F. et al, 1981). Another study (Strobl, W. et al, 1983) determined the levels of apolipoproteins A-I, AII, B and E, as well as total cholesterol and triglycerides in cord serum and capillary serum from infants in the first week of life. The study set out to deter­mine the cord serum lipoproteins. It was found that the serum apolipoproteins A-II, and B were lower at birth than in adulthood. Interestingly it was also seen that apoE showed no difference between neonates and adult levels. Furthermore, during the first five days of life the apoB levels more than doubled whilst apoA-I increased moderately. ApoE also showed a moderate rise whereas apoA-II showed no significant change. The study’s conclusion was that the “…changes of the apolipoprotein pattern during the first week of life reflect the evolution of the lipid transport system.” (Strobl, W. et al. 1983). Of interest here is that the Strobl study shows that apoproteins can be detected early – a useful tool to detect early type IV?

A proposal for further research.

It has been shown that in rare instances hypertriglyceridaemia can be caused by a structural gene mutation affecting lipoprotein lipase or apolipoprotein C-II (Galton, D. J. et al, 1982). In the majority of type IV cases no mutation is detectable. This raises the possibility again that a polygenic mechanism is involved. It is thus worthwhile to thus ^“…consider the apoprotein genes as one of the genetic factors determining the common forms of hypertriglyceridaemia.” (Rees, A. et al, 1983). The Rees study was to detect a DNA polymorphism adjacent to the human apoprotein gene isolated earlier (Shoulders, C. C. et al, 1982) and its relation to type IV familial hypertriglyceridaemia.

A research requires subjects and this in the case of hypertriglyceri­daemia implies a register of patients. The main aim would be to determine the levels of apoproteinin young or pre-adult age groupsloosely related to VLDL. As we have seen, VLDL contains the major apoproteins B, C-I, C-II, C-III and E (Kane, J. P. et al, 1975). Tests would have to be directed firstly to the offspring and siblings (and their offspring) of known patients with type IV hyperlipoproteinaemia. The tested siblings for associated VLDL apoproteins, as well as tested offspring, would depend on the age of the proband. The central theme is that where standard lipid level tests are inconclusive in adolescent (and younger) offspring of type IV patients, it may be possible to determine apoprotein levels indicative of type IV in the pre-clinical phase.

Methods employed must go beyond, as already stated, the standard lipid plasma level tests. Techniques could include the use of labelled apoproteins with radioactive iodine complexed to lipoproteins and injected into the subjects to be tested. Such a method was used (Fisher, A. et al, 1982) to study familiaI type III “…associated^–with^, a particular isoform of apolipoproteim E ^.(apoE) called^–E-2.”  As is known apoE is an important constituent of chylomicrons and VLDL remnants. It was seen that the amino-acid sequences of E-2, E-1 and E-4 were identical except at two sites (A and B) and it was further shown that “Arginine residues seem to be essential for proper lipoprotein-receptor binding in general…” (Fisher, A. et al, 1982). The techniques of isoelectric focusing and 2-D electrophoresis were used, and the results from each method compared, to determine a variant of apoA-I (Schamaun, 0.  et al, 1983). The variant isolated was termed apoA-I 2-1 and established as a co-dominant autosomal Mendelian inheritance. Despite its not being associated with any disease process the conclusion was that the apoprotein “…may be regarded as a valuable marker for genetic mapping purposes”. Isoelectric focusing of lipoprotein VLDL was employed to apoE phenotype type V hyperlipidaemic patients (Stuyt, F. M. J. et al, 1982). All the patients involved had severe hypertriglyceridaemia with 10 mmo1/1 or more. It was found that none of the “…twenty type V patients had the homozygous E-4 phenotype and eight had the heterozygous E-4 phenotype…apoE allele frequency was similar in the control group and in the type V patients.

It seems unlikely that apoE-4 is a major determining factor in the expression of the disorder.” This may be contrasted with phenotypes determined by gel isoelectro-focusing used elsewhere (Ghiselli, G. et al, 1982). It was pqinted out that apolipoprotein E (apoE) may be inherited at a single locus with three common alleles (E-3, E-2, and E-4). The products of these alleles are termed apoE-2, apoE-3, and apoE-4. Results showed that patients with types I, IIa, IIb, and IV hyperlipoproteinaemias had a similar apoE- phenotype distribution similar to normal with 40 to 60„, homozygous for E-3. The conclusion drawn was that apoE-2 and apoE-4 are associated with two distinctly different dyslipoproteinaemias and that apoE has also two different physio­logical functions. More sophisticated techniques were employed for genetic analysis (Rees, A. et al, 1983) using appropriate recombinant DNA plasmid probes. The purpose was to detect variations “…of the genotype…by comp­arison of characteristic genomic patterns of recombinat DNA.” Rees A et al stated that they used techniques of digestion with restriction endonucleases and the Southern blot hybridisation method for their nuclear whole blood pellets (Baralle, F. E. et al, 1980; Southern, E. M. 1975).

There exist techniques for the determination of the apoproteins that are associated with VLDL and especially with type IV hypertriglyceridaemia. It is therefore suggested that if these techniques were applied to the offspring, siblings, and immediate relatives of known type IV patients, that it may be possible to detect those children and adolescents liable to develop clinical hypertriglyceridaemia later in life – especially before standard plasma lipid level determinations become an effective diagnostic tool during adult life.

Summary and conclusions.

Analyses of serum lipid cholesterol and triglycerides have been carried out. One such programme determined these levels on an autoanalyzer (Cresanta, J. L. et al, 1983) using a random sample of the population. The difference is that the register based research avenue suggested in this essay would not be random because subjects would be selected on the basis of their relationship to a known hypertriglyceridaemic proband. However, Cresanta et al found that the “Mean beta-LPC declined during early adolescence…as did the adolescent decline in mean total cholesterol, while mean pre-beta-LPC increased during the same period as did the early adolescent increase in mean triglycerides.” (my italics).

In conclusion it is worthwhile saying that research into the earlier detection of hypertriglyceridaemia (as well as the other types of hyper­lipoproteinaemia) is a contribution to the development of preventive cardiology. By concentrating on the younger age groups it is intended to try and raise the diagnostic capabilities for type IV to the level at present available for hypercholesterolaemia in younger age groups.- One important study^,: “…examined the hypothesis that familial aggregation of lipids and lipoproteins facilitates within-family identification and hyperlipoproteinamia.” Using this model and applying it to a lipid register it may be possible to extend apoprotein techniques of determination into known family groups. As the above study pointed out “As the categorizalion of probands hypercholesterolaemia or hypertriglyceridaemia increased from sporadic, to persistent, to severe, the percentage of hypercholesterolaemic or hypertriglyceridaemic offspring and siblings increased.” Furthermore, and to a certain extent relevant to family entries on lipid registers₎it can be shown that “Close sibling and parent-offspring lipid and lipoprotein risk factor associations in hypercholesterolaemic and hypertriglyceridaemic family units during and after the period of shared common-household environment facilitate within-family identification of dyslipoproteinaemia and suggest potential sharing of coronary heart disease risk.” (Morrison, J. A. et al, 1983).

Such a conclusion posits not only the value of further lipid gene and apoprotein detection within at risk families for CHD,it also hints at family orientated dietary changes – especially when that family contains one or more diagnosed hyperlipoproteinaemic patients. However, thisimportant preventive avenue is beyond the scope of this present essay which has to concentrate on its set taskand that is to add to preventive cardiology by elucidating as soon as possible all individuals who later in life may be at risk from coronary heart disease.

Eric .W. Edwards March 16th, 1984.

Essay contribution to the Simon Broome Heart Disease Research Trust.

References.

 Alaupovic P.  Conceptual development of the classification systems of plasma lipoproteins. in Peeters H (ed), Protides of the Biological; Fluids. Pergamon, Oxford, 1971.

Alaupovic P, Lee DM, McConathy WJ. Studies on the composition and structure of plasma lipoproteins: Distribution of lipoprotein families in major density classes of normal human plasma lipoproteins. Biochim Biophys Acta 260 689-707 1972

Albers JJ, Cabana VG, Hazzard WR. Immunoassay of human plasma apolipoprotein B Metabolism 24 1339 1975

Avogaro P, Bittolo Bon G, Cazzolato G, Quinci GB, Belussi F  Plasma levels of apolipoprotein A, and B in human atherosclerosis Artery 4 385-94 1978

Avogaro P, Bittolo Bon G, Cazzolato G, Quinci GB. Are apolipoproteins better discriminators than lipids for atherosclerosis? Artery I 901-3 1979

Baralle FE, Shoulders CC, Goodburn S, Jeffreys A, Proudfoot NJ  The 5′ flanking region of human epsilon-globin gene Nucleic Acid Res. 8 4393-404 1980

Beckenridge WC, Little JA, Steiner G, Chow A, Poapst M  Hypertriglyceridaemia associated with deficiency of apolipoprotein C-II New Eng. J. Med. 298 1265 1978

Biesiegel U, Utermann G. An apolipoprotein analog of rat apolipoprotein A-IV in human plasma Fur. J. Biochem. 93 601 1979

Blanchette-Mackie EJ, Scow RO. Effects of lipoprotein lipase on the structure of chylomicrons J. Cell Biol. 58 689-708 1973

Blum CB, Aron L, Sciacca R. Radioimmunoassay studies of human apolipoprotein E J. Clin. Invest. 66 1240 1980

Brewer HB jr, Lux SE, Ronan R, John KM. Amino-acid sequence of human apoLp-GLn-II (apoA-II), an apolipoprotein isolated from the high density lipoprotein complex

Proc. Nat. Acad. Sci. USA 69 1304 1972

Brewer HB jr, Shulman R, Herbert P et al. The complete amino-acid sequence of alanine apolipoprotein (apoC-III), an apolipoprotein from human plasma very low density lipoproteins J. Biol. Chem 249(15) 4975-4984 1974

Brewer HB jr. Current concepts of the molecular structure and metabolism of human apolipoproteins and lipoproteins. Klin. Mochenschr. 59(3) 1023-1035 1981

Catapano AL, The distribution of apoC-II and apoC-III in very low density lipoproteins of normal and type IV subjects. Atherosclerosis 35 419 1980

Cheung MC, Albers JJ. The measurement of apolipoprotein A-I and A-II levels in men and women by immunoassay. J. Clin. Invest. 60 43 1977

Cresanta JL, Srinivasan SR, Foster TA, Webber LS, Berenson GE  Distributions of serum lipoproteins in children by repeated measurements Prev. Med. 12 554-568 1983

Curry MD, McConathy WJ, Fesmire JD, Alaupovic P, Quantitative determination of human apolipoprotein C-III by electro-­immune-assay. Biochim Biophys Acta 617 503 1980

Fisher A, Gahl WA. Cysteamine in treatment of type 3 hyperlipidaemia Lancet 2 1131 1982

Fredrickson DS, Goldstein JL, Brown MS. The Familial Hyperlipidaemias., in The Metabolic Basis of Inherited Disease, 4th Edition, eds Stanbury JB, Wyngaarden JB, Fredrickson DS. NY, 1978

Galton DJ, Stocks J. Dodson PM. Lipoproteins: their role in enzyme regulation Clin. Biochem. Rev. 3 377-405 1982

Ghiselli G, Gregg R, Schaefer EJ, Zech LA, Brewer BH. Phenotype study of apolipoprotein E isoforms in hyperlipoproteinaemia patients Lancet 2 405 1982

Goldstein JL, Ho YK, Basu SK et al

Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc. Nat. Acad. Sci. USA 76(1) 333-337 1979

Havel RJ, Kane JP, Kashyup ML. Interchange of apolipoproteins between chylomicrons and high density lipo­proteins during alimentary lipaemia in man. J. Clin. Invest. 52 32-38 1973

Havel RJ, Kotite L. Kane JP. Isoelectric heterogeneity of cofactor protein for lipoprotein lipase in human blood plasma. Biochem Med 21 121-28 1979

Havel RJ, Chao YS, Windier EE, Kotite L, Guo LSS. Isoprotein specificity in the hepatic uptake of apolipoprotein E and the pathogenesis of familial dys-betalipoproteinaemia

Proc. Nat. Acad. Sci. USA 77 4349 1980

Herbert PN, Assmann G, Gotto AM jr, Fredrickson DS. Disorders of Lipoproteins and Lipid Metabolism., in The Metabolic Basis of Inherited Disease, 5th Edition. Eds Stanbury JB, Wyngaarden JB,

Goldstein JL, and Brown MS. McGraw Hill, NY, 1983.

Higgins JM, Fielding CJ. Lipoprotein lipase: mechanism of formation of triglyceride rich remnant particles from very low density lipoproteins and chylomicrons. Biochemistry 14(11) 2288-2292 1975

Jackson RL, Sparrow JT, Baker HN, Morissett J, Taunton OD, Gotto AM jr  The primary structure of apolipoprotein-serine .5^–2Biol. Chem 249 5308 1974

Jackson RL, Baker N, Gilliam EB et al. Primary structure of very low density apolipoprotein C -II of human plasma Proc.Nat.Acad.Sci.USA 74(5) 1942-45 1977a

Jackson RL, Morrisett JD, Gotto AM jr. Lipoproteins and Lipid Transport: Structural and Functional Concepts., in: Hyperlipidaemia, Diagnosis and Therapy, eds Levy RI and Rifkind BM, Grune and Stratton, NY, 1977b

Kane JP, Sata T, Hamilton RL, Havel RJ. Apoprotein composition of very low density lipoproteins of human serum J.Clin.Invest 56 1627-34 1975

Kane JP, Hardman DA, Paulus HE. Heterogeneity of apoprotein B: isolation of a new species from human chylomicrons Proc.Nat.Acad.Sci.USA 77 5923 1980

Kashyap ML, Srivastava LS, Chen CY, Perisutti G, Campbell M, Lutmer RF, Glueck CJ  Radioimmunoassay of human apolipoprotein C-II. A study in normal and hyper­triglyceridaemic subjects. J.Clin.Invest 60 171 1977

Kostner GM. Studies of the composition and structure of human serum lipoproteins. Isolation and partial characterization of apolipoprotein A-III. Biochim Biophys Acta 336 383-94 1974

Lim CT, Chung J, Kayden HJ, Scanu AM. Apoproteins of human serum high density lipoproteins. Isolation and characterization of the peptides of Sephadex fraction V from normal

subjects and patients with abetalipoproteinaemia. Biochim Biophys Acta 619 129 1980

Mahley RW, Innerarity TL, Brown MS et al. Cholesteryl ester synthesis in macrophages: Stimulation by beta-very low density lipoproteins from cholesterol-fed animals of several species J.Lipid Res 21 970-980 1980

Mao SJT, Sparrow JT, Gilliam.EB, Gotta AM jr, Jackson RL. Mechanism of lipid-protein interaction in the plasma lipoproteins: lipid binding properties of synthetic fragments of apolipoprotein A-II Biochemistry 16 4150 1970

McConathy WJ, Alaupovic P. Isolation and partial characterization of apolipoprotein D: A new protein moiety of the human plasma lipoprotein system. FEBBS Lett 37 178 1973

Morrison JA, Namboodiri K, Green P, Martin J, Glueck CJ. Familial aggregation of lipids and lipoproteins and early indentification of dyslipoproteinaemia. JAMA 250 14 1983

Murphy EA, Kwiterovich PO. Genetics of the Hyperlipidaemias., in The Hyperlipidaemias., eds Rifkind BM and Levy RI, 1977

Olofsson SO, McConathy WJ, Alaupovic P. Isolation and partial characterization of a new acidic apolipoprotein (apolipoprotein F) from high density lipoproteins of human plasma. Biochemistry 17 1032 1978

Rall SC jr, Weisgraber KH, Mahley RW. Human apolipoprotein E – the complete amino-acid sequence J.Biol.Chem 257(8) 4171-78 1982

Rees A, Shoulders CC, Stocks J, Galton DJ, Baralle FE. DNA polymorphism adjacent to human apoprotein AI gene: Relation to hypertriglyceridaemia. Lancet 1 444 1983

Schamaun 0, Olaisen B, Gedde-Dahl T jr, Teisberg P  Genetic studies of an apoA-I lipoprotein variant Hum.Genet 64 380-83 1983

Schonfeld G, Lees RS, George PK, Pfleger B. Assay of total plasma apolipoprotein B concentration^–in human subjects J.Clin.Invest 53 1458 1974

Schonfeld G. Disorders of lipid transport – update 1983 Prog.Cardiovasc.Dis XXVI/2 Sep-Oct 1983

Segrest JP, Jackson RL, Morrisett JD, Gotto AM jr. A molecular theory of lipid protein interactions in the plasma lipoproteins FEBS Lett. 38 247 1974

Shepherd J, Bickers S, Lorimer AR, Packard CJ  Receptor-mediated low density lipoprotein catabolism in man J.Lipid Res. 20 999 1979

Shepherd J, Packard CJ, Bickers S, Lawrie TDV, Morgan HG. Cholestyramine promotes receptor-mediated low density lipoprotein catabolism N.Eng.J.Med 302 1219 1980

Shore VG, Shore G. Heterogeneity of human plasma very low density lipoproteins: Separation of species differing in protein components. Biochemistry 12 502 1973

Shoulders CC, Baralle FE. Isolation of the human HDL apoprotein A-I gene Nucleic Acid Res. 10 4873-82 1982

Shulman RS, Herbert PN, Fredrickson DS, Wehrly K, Brewer HB jr. Isolation and alignment of the tryptic peptides of alanine apolipoprotein, an apolipoprotein from human plasma very low density lipoprotein J.Biol.Chem 249 4969 1974

Shulman RS, Herbert PN, Wehrly K et al. The complete amino-acid sequence of C-I (apoLp-Ser), and^,apolipoprotein from human very low density lipoproteins. J.Biol.Chem 259(1) 182-90 1975

Simons LA, Gibson JC. Lipids: A Clinicians Guide. MTP Press Ltd, Lancaster, 1980

Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J.Mol.Biol. 98 503-17 1975

Sparrow JT, Morrisett JD, Pownall HJ, Jackson RL, Gotto AM jr. The mechanism of lipid binding by plasma lipid proteins: Synthesis of model peptides in: Walter R and Meinhofer J(eds), Peptides: Chemistry, Structure, Biology. Ann Arbor, Michigan, Ann Arbor Science, 1975

Steiner G. Reardon MF. A new model of human VLDL metabolism based on simultaneous studies of its apoB and triglyceride. Metabolism 32(4) 342-7 1983

Strobl W, Widhalm IC, Kostner G, Pollak A. Serum apolipoproteins and lipoprotein (a) during the first week of life Acta Paed Scand 72(4) 505-509 1983

Stryer L. Biochemistry. Freeman, USA, England 1974

Stuyt PMJ, Stalenhoef AFH, Demacher PNM, Van’t Laar A Hyperlipoproteinaemia type 5 and apolipoprotein E4 Lancet 2 934 1982

Steger T, Fex G. Apolipoprotein A-I and B levels in adolescents; a trial to define subjects at risk for coronary heart disease. Acta Paed Scand 72 499-504 1983

Vaith P, Assmann G, Uhlenbruck G. Characterization of oligosaccharide side chain of apolipoprotein C -III from human plasma very low density lipoproteins

Biochim Biophys Acta 541 234-240 1978

Whayne TF, Alaupovic P, Curry MD, Loe ET, Anderson PS, Schechter E. Plasma apolipoprotein B and VLDL-, LDL-, and HDL- cholesterol as risk factors in the development of coronary artery disease in male patients examined by angiography. Atherosclerosis 39 411-24 1981

Zannis VI, Breslow JI. Human very low density lipoprotein E isoprotein polymorphism is explained by genetic variation and post translational modification. Biochemistry 20(4) 1033-1041 1981

 

HDL levels and CHD in Oxfordshire

Pressure by Oxfordshire GP’s for screening facilities for high density lipoprotein (HDL) is a welcome development. Unfortunately the predictive ability of HDL alone is not however accurate enough in coronary heart disease (CHD) risk estimation. True an inverse relation between HDL and CHD has been confirmed by prospective studies and subsequently shown to be independent of other risk factors.

The usual HDL blood level is between 1.1 and 1.7 mmols with raised levels found in athletes and pre-menopausal women – higher than average levels regarded as protective against CHD. Levels below 1.0 are predictive of increased susceptibility to CHD. HDL levels relate negatively to degree of obesity and show a positive correlation with exercise participation, physical fitness and alcohol consumption. Plasma HDL tends to be low amongst the over-weight, sedentary individuals and smokers. The Kilkenny Health Study (1985) detected amongst males and females the following HDL levels:

Age            Male             Female

35-44         0.91                1.13

45-54         0.90               1.13

55-64         0.94                1.13

Age-sex related HDL levels.

Kilkenny Heart Study, 1985.

The Heartbeat Wales Survey (1985) showed that HDL levels fall with increasing age, with female levels higher at all ages. An Israeli study (1985) showed that age adjusted mortality increased with decreasing HDL levels. HDL therefore provides an additional tool for assessment of CHD risk with, according to the British Regional Heart Study (Pocock, S. J. 1986), a one standard deviation increase in HDL indicating a 10% decrease in risk, whereas a one standard deviation increase in non-HDL (TC-HDL) indicating a 50% increase in risk. Further estimations (Pocock, S. J. 1989) calculated a 2.5% reduction in IHD with every 10mg/L (0.026 mmol) of HDL elevation.

Most importantly though determination of HDL enables us to calculate the low density lipoprotein (LDL) component – the major atherogenic particle of total cholesterol (TC). Measurements of these cholesterol fractions can be found by knowing two of them from the following formula: LDL = TC – TG/5 – HDL (in mmols). This is useful because studies of CHD risk assessment emphasises the predictive power of the LDL:HDL ratio. LDL can be calculated using the Friedwald Formula (FF) of TC – HDL – (TG x 0.45) in mmols or in another form: LDL = TC – HDL – TG/2.19 (mmols).

Does estimation of HDL cholesterol subfractions imply the impending redundancy of serum TC or other lipoprotein fractions? The answer must be no. Even though serum TC is only a first approximation requiring serial validation a generalised TC screening procedure is still required. This is because any population strategy for CHD risk estimation must encompass the determination of as complete a lipid profile as possible.

Serum triglycerides  (TG) have been shown to predict IHD risk in men with low serum TC (= < 220 mg/100 mls) and the Framingham Study viewed TG as a risk factor if the TC to HDL ratio exceeded 3.5. In this respect calculation of LDL is more precise for plasma with TG concentrations within normal range using De Long’s modification of the original FF thus: LDL = TC – (HDL + 0.37 x TG) for mmols (0.16 x TG for mg/100 mls). In order to calculate LDL using an FF that varies with TG it is better to use Rao’s modification thus: LDL = TC – HDL -TG (0,47 – (0.022 x TG). LDL estimates of risk based on recent transatlantic concensus suggest that with hypercholesterolaemia as the only risk factor it is advisable to reduce LDL to below 4.9 mmol. With hypercholesterolaemia plus two other risk facors an LDL level of 4.1 mmol was recommended.

Hyperlipidaemia has been described as serum TC > 7.2 mmol and/or serum TG > 2.0 mmol – though caution has to be exercised here because of population variation. For example the estimated mean TC in eastern Finland (a high CHD country) is 270mg/100 mls (6.98 mmol), whereas Japan (a low CHD country) has an estimated mean TC of only 170mg/mls (4.39 mmol). In addition, even though there is disagreement about TG’s as independent risk factors for CHD, hypertriglyceridaemia in the presence of low HDL concentration is possibly associated with increased vascular disease (hypertriglyceridaemia = > 2.00 mmol, HDL = < 0.9 mmol).

The complexity of inter-population and intra-population variation in lipid profiles is further compounded by intra-individual variation. For  example it has been shown that TC shows temporal variations over a six hour period – from a nadir of 189 mg/100 ml (4.26 mmol) between 10 and 11 am to a zenith of 211 mg/100 ml (5.45 mmol) between 3 and 4 pm, with a periodic cycle of 50 minutes. How often are samples taken in surgeries and clinics during morning hours? Such diurnal and circadian variations shown by lipid levels must be noted when determining lipid profiles in both the individual patient and in populations.

Despite the arbitrariness of cut-points guidelines must remain for assessment of serum lipid parameters. The National Institutes of Health (USA) determined the following groups in mmols:

Age          Acceptable     Moderate Risk     High Risk

20-29      > 5.17              5.18 – 5.68             < 5.69

30-39      > 5.69             5.10 – 6.20             < 6.21

40 +        > 6.21              6.22 – 6.71             < 6.72

National Institutes of Health, USA  (in mmols).

MONICA (monitoring trends in coronary heart disease study) in Scotland settled on the following cut-points by age and sex for median TC levels in 10 year  groups, in mmols:

Age           Men        Women

23-34        5.5             5.2

35-44        6.0            5.5

45-54        6.3            6.4

55-64        6.2            7.2

MONICA, Scotland

10 year cut-points by sex for median TC.

The European Atherosclerosis Society guidelines suggested 5.2 mmol as acceptable. 5.2 to 6.5 mmol required dietary intervention, 6.5 to 7.8 mmol required lipid lowering agents and < 7.8 mmol needed referral to a specialist lipid clinic. The British Four Centres Sudy (Mann, J. I. 1988) used cut-points for TC of 5.5, 6.5, and 8.0 mmol with over 6.5 indicating clinical care – the mean male and female blood TC in Oxford of 5.8 mmol were above the World Health Organisation optimum level of 5.17 mmol.

High risk emerges above the 75th percentile for TC and accounts for 40 to 50% of CHD cases, but 50 to 60% of CHD cases appear in the 75% of the population below the 75th percentile (with familial hypercholesterolaemia at the 99th perecentile) – and obvious case for more accurate lipid analysis that serum TC measurements that ignore HDL and the atherogenic LDL component. For example a case study from the Cleveland Clinic Foundation of a woman athlete with elevated TC was considered as a moderate risk using 75th percentile measurements. Further tests showed an elevated HDL that reduced her LDL to safe limits. Again this emphasises the validity of partitioning TC into atherogenic LDL and anti-atherogenic HDL fractions.

In addition, estimation of CHD susceptibility utilising serum cholesterol presents problems because the relation of TC to CHD incidence becomes attenuated with advancing age. The Framingham Study has shown that beyond the age 55 TC levels can no longer predict CHD. However, with fractionation into lipoprotein components (LDL, HDL, VLDL) the relationship of the lipid profile to CHD re-emerges. This is particularly of value in assessing risk in those individuals susceptible to premature CHD in age groups 45 to 64. Likelihood ratios for men (TC 1.98, HDL -14.0, LDL  4.4, VLDL  1.03) and women (TC 2.26, HDL -21.2, LDL 4.5, VLDL 5.04) represent predictive strengths for the 50 to 80 years group, A number of ratios for likelihood risk for both sexes aged 50 to 80, that include LDL:HDL ratios, have been calculated for expectancy in both normo- and hyerlipidaemic individuals.

A valuable predictive model once HDL and LDL values are known is the estimation of the Cholesterol Retention Fraction (CRF) used in the Bowling Green Study (USA). This formula has not, as far as is known, ever been applied to any British data. The only use of CRF in Britain at present is being applied, by the present author and researcher, to data from the Heartbeat Wales Study to which the author has access. The CRF is derived from (LDL – HDL)/LDL which gives the following predictor values:

Predictor values

>  0.70  ———->  atheroscerosis

<  0.69  ———->  mitigates CHD

<  0.60  ———->  implies immunity

CRF applied to Heartbeat Wales data.

The accuracy of CRF depends upon the proviso that LDL concentration is < 4.4 mmol or LDL is 1.05 mmol and that systolic blood pressure is < 140 mm Hg. Here we have lipid predictors linked to BP – a much more enhanced model for individual and population estimation of CHD risk and susceptibility.

HDL assessment should be part of a wider population prevention strategy, an approach that attempts to determine population attributable risks for a number of risk factors in all age groups – especially the young. This was the main aim of the ill-fated proposed project titled the Oxford City Heart Health Initiative which received no applied for national or local funding. The estimation of HDL does not provide a key to open all locks, but it does give GP’s a more precise tool to estimate lipid profiles and correlate them with other risk factors for CHD susceptibility in their patients.

[An unpublished reply to an article in the Oxford Times. Submitted 30.6.1990, while author was engaged on university research into the anthropology and genetics of CHD risk factors including HDL, LDL and total cholesterol].

Maritime Anthropology of the Manila Galleon Trade: a proposed investigation

1. Historical Introduction

The Philippine Islands were colonised from Mexico (New Spain) in 1564 (Hayes, 1934), and it was after Legazpi’s voyage that Mexico’s contribution to the Philippines became the Acapulco-Manila galleon trade (Perez, 1954), ships now described as veritable argosies of cargo and treasure.

Historically  what became known as the Sisterhood of Manila and Acapulco began in October 1565 when Andres de Urdeneta navigated Legazpi’s San Pedro safely back to Acapulco (Zaide, 1971). Thus “It was  New Spain or Mexico that contributed most directly to the extraordinarily rapid growth of Manila as the pearl of the Orient…” (Perez, 1954). Manila was best geographically suited for drawing together Chines and Japanese silk, Moluccan spices, Indian cottons and Cambodian ivory which were funnelled to the argentiferous Spanish colonies of the New World (Legarda, 1955).

The golden age of Manila and Acapulco, especially during the seventeenth and eighteenth centuries, was based on the famous galleon trade (Zaide, 1971). It has been said that (Rodriguez, 1941) during 250 years (1565-1815) of the Manila-Acapulco trade some 108 government owned galleons voyaged across the Pacific and ferried “…thousands of men and many millions of treasure.” The Manila-Acapulco service was as old as the Spanish colonisation of the Philippines and bound up with the very life of the islands – the galleon trade was the umbilical cord upon which the colony’s existence depended. The arrival of the galleons in Manila was met with rejoicing and meant a year of prosperity, whereas loss by shipwreck or piracy meant a year of economic depression (Perez, 1954). Thus “En los doscientos cincuenta anos de existiencia del galleon, solamente naufragarion 30, calculados en unos 1,600 hombres los desaparecidos y valorandose las perdidas de las mercancias en 6o millones de pesos.” (Lorente Rodriguez, 1944). Thus “The failure of the Philippine galleon to arrive causes a scarcity of many things in this country.” (Croix, 1749).

Annually the Manila galleons (Naos de China) crossed the Pacific with valuable cargoes (Zaide, 1971) and used to ship expensive luxury commodities in both directions, the monetary value of their cargoes was enormous (Ives, 1964). The vice-royalty of New Spain was the principal market for the Manila galleon cargoes (Schurz, 1918). A new era began in the islands history with the establishment of direct trading with China (Legarda, 1944) with Chinese traders from the Celestial Empire arriving in 1572 (though they had traded there for years before the Spanish arrival). Most important of all the Acapulco bound goods was Chinese silk (Schurz, 1939).

Ships from the Asian countries arrived and eventually increased the diversity of nationalities and ethnic groups in Manila (Legarda, 1944), the goods brought by these traders (and then eastward as galleon cargoes comprised Persian rugs, Indian cottons, ivory, jasper, jade, copper, brass, spices, musk, borax, japanned boxes, inlaid escritoires, lead, camphor, porcelain, earthenware, pearls, precious stones, etc. Philippine products were gold dust, wax, cordage, blankets, sail cloth, linen, etc. From Acapulco there came chests of Mexican and Peruvian silver, all the artefacts and necessities of the colonial administration, merchants, administrators, missionaries and clerics (Jesuits, Franciscans, Dominicans, Augustinians), soldiers and officials. This arrangement kept the Philippines tied to the apron strings of Mexico (Le Roy, 1905) with Spanish money in the earlier years not for the benefit of the islands but for the Spanish expansion into the far east. Manila was a jumping off point for expeditions. No permission was however given to legitimate emigrants unless they agreed to become a citizen of the colony (Schurz, 1918).

Numerous Chinese came yearly in junks and sampans and added to the burgeoning population of Manila (Lagarda, 1944) and the silver they received from the Manila galleons was taken back in large quantities to China. As Boxer (1970) pointed out “Many foreign merchants and travellers in Asia, Portuguese, Spaniards, Dutch, English and French alike, commented on the extraordinary demand for silver in India and China during the sixteenth and seventeenth centuries.” especially the Chinese demand for Spanish pesos de ocho reales. This situation was also noted by Morga (1609) and Santos (1609) in Manila, and again recalled by Manrique (1649) after a visit there between 1637 and 1638. Mexican silver reaching Manila was just as eagerly sought by other neighbouring peoples (Diaz, 1890).

Attracted to the profits of the galleon trade the Spaniards in the Philippines neglected the agricultural and industrial development of the islands (Bourne, 1907) with the land remaining predominantly in the hands of the indigenous owners. Large tracts of land were held by religious orders and half-caste Chinese who achieved this via their control of internal trade and small credit (Schurz, 1918; Bourne, 1907). Internal trade therefore passed to the hands of non-Spaniards. Locally the “Filipinos’ were unprepared by their previous history for the whirligig of events into which Spain’s conquest plunged them.” (Legarda, 1944).

The cargo of the Manila galleon was distributed very widely throughout Spanish America with imports into Acapulco reaching as far as Peru, Guatemala, Campeche, Caracas, the Windwards and the Greater Antilles (Schurz, 1918). Even today “…Mexican senoritas during festive occasions wear the China poblana dress, whose red, green, and white colors have become the colors of Mexico’s national flg.” (Zaide, 1971), an echo of the ramifications and effects of the silk trade brought by the galleons from Manila.

2.  Context of the Research

The Manila “…galleon trade was the product of policies that have to be looked at from an overall point of view if they are to be understood in their proper perspective.”  The effects, successes and losses, of the galleon trade require a similar overall perspective, not just the successful voyages, but also the unsuccessful. The attitudes and problems that surround the galleon trade and the population of the islands provide a key to  unravelling the colonial history of the Philippines, the trans-pacific trade, the south east Asia commerce, and the population diversity of the Philippines. The galleon trade can be seen as a series of time-capsules whose documentary evidence contains an invaluable insight and means to understand the development of the population of the Philippines, as well as trans-Pacific population movements.

Ships do not sail in a cultural vacuum (Lenihan, 1986) which implies that the galleons were a material expression of a wider cultural dynamic. The galleon trade lasted 25o years and provides an enormous documentary resource for the study of human maritime and cross-cultural activity. Moreover, a galleon can be seen a “…a cultural component that shares some conventions with the parent culture, it is also a cultural entity in and of itself.” (Murphy, 1986). Manila-Acapulco galleons in this context can be seen as microcosms of inter-relating cultures, functioning not in the archaeological sense as time-capsules fixed in time, but anthropologically as vectors of human activity.An anthropological perspective of the galleons themselves must include recognition of their self-sufficiency as maintained shipboard communities which were at sea for months before landfall (Lenihan, 1986). In addition other aspects include on-board stress factors, diseases, malnutrition, hierarchical shipboard society, always composed of several nationalities and strata. A galleon and its material culture had a narrowly defined purpose. Whereas with regard to the effects of the galleon trade a wider maritime anthropological perspective would recognise the important implications for our “…knowledge of global communications dynamics.” (Watson, 1986), our knowledge of the networks formed and developed, how they functioned and “…especially what the mutual effects of contact were on the societies in question.”. The galleons not only carried out and represented aspects of their parent culture they also provide important perspectives on social processes, primary vectors in an exchange system (Murphy, 1986).

3.  Objectives of the Research

The research programme will investigate the effects of the galleon trade recognising that Manila and Acapulco “…from the later part of the 16th to the beginning of the 19th century…played a significant role in history…” (Zaide, 1971), especially that of the Philippines. The development of the peoples of the Philippines is underpinned by the galleon trade, and the history of the ethnic population structure of those islands was to a large extent moulded by the galleon trade and the commerce that it generated with south east Asia. The galleon trade suffered many shipwrecks and losses which because of their intrinsic value exerted some considerable effect on the well-being of the colony. The ships themselves were transporters of people, not just silver,  and in both directions. The investigation of the galleons and their losses and successes opens up avenues to explore the cultural and population changes that occurred in the islands during the 16th and 17th centuries, as well as illuminating the links between Mexico and the other side of the Pacific.

The object of the research and its written papers will be to investigate and disseminate knowledge of the Manila Galleon trade and its effects on the population dynamics of the Philippines, its ramifications with South East Asia (as shown by the movement of cargoes and people in the Introduction above). In so doing the aim will be to develop a perspective of historical maritime anthropology.

The analysis of documentation relating to cargo and people on board (crew, religious, merchants, soldiers, migrants, officials) will thus contribute much to the reconstruction of the rapidly changing social, cultural and population structures of the societies involved. The research will explore questions pertaining to the contrasts of maritime culture relating to point of origin and destination, e.g., Chinese, Japanese, Spanish, Dutch and the indigenous population. Much of the historical documentation to be studies was recorded by persons who were compelled by authority to do so. These materials often reflect attitudes of the authority group and attention will be paid to the conflicting views of the Spanish, Dutch and Chines. Study of documentary materials that surround the galleon trade will clarify the variability in human activities it engendered.

A maritime anthropological perspective of the galleon trade implies developing a cross-cultural framework which juxtaposes traits and trait complexes, or various aspects of cultural systems from widely separated geographical proveniences. The galleon trade and its effects can be seen as an anthropological phenomenon. The associated documentation provides a unique resource containing important information and ideas concerning human maritime, social and cultural behaviour. The main hypothesis to be tested is that the Manila galleon trade, as a maritime anthropological phenomenon, played a significant role in determining the population structure and dynamics if the Philippine Islands, and commercial development in South East Asia.

4.  Significance of the Research

The project will aim to develop a perspective of historical maritime anthropology with special reference to the effects of the galleon trade. Most of the archival material to be studies has not been analysed to date or referred to in previous literature. This will comprise an innovative and unique feature of the research.

The significance of the galleons is that they were integral aspects of their larger parent culture, the culture on board a galleon considered as a specialised statement of the way a society relates to other societies (Murphy, 1986). The galleons were closed communities which were opened at both ends of the voyages. The study of the galleon trade can significantly contribute much to our understanding of human activities in many areas. The trade and its associated documentation affords an insight into social and cultural relationships over space and time. The cargoes of the galleons were significant indicators of the parent culture’s priorities in view of overseas and home involvements. The galleon cargoes are also highly informative on the activities of the different populations and cultures involved in the trade.

The galleons in effect became vectors for the spread of people, technology, beliefs, with diffusion of social and cultural traits. A study of the trans-Pacific galleon trade and Spanish colonising process is significant in that it has great potential to improve our “…understanding of global communication networks.” (Watson, 1986), as well as the resulting cross-cultural exchanges that developed. The research will aim to identify consistent and reliable relationships between particular kinds of human activity with the galleon trade as the linking factor (Gould, 1986). This will make it possible to posit generalisations concerning past human maritime activities, to develop the field of historical maritime anthropology via the galleon trade.

5.  Research Methods

The research will entail obtaining, translating and analysing copies of original 1th, 17th and 18th century documents primarily held by the Archivo General de Indias in Seville, secondarily by Archives in Madrid, Manila, Amsterdam, Paris, and Barcelona. Other material sources are the Bodleian Library, British Library and numerous other archival resources. Such documents and materials will be recorded on a computer database. The senior researcher is a registered reader with Seville, Madrid (including the Museo Naval) Paris, and Amsterdam archives. The Seville research assistant is registered with the Archivo General there. The research will lead to the creation of and continuing maintenance of a large bibliographic database.

The research intends to write papers for publication and dissemination. Copies of all documents obtained so far and id in future will be held as a special collection in the designated Oxford college. The computer database and bibliographies will also be held in the library. Such materials to be made available for use by other scholars and researchers.

6.  References and Sources Consulted

Bourne, E. G.  (1907).  Discovery, Conquest and Early History of the Philippine Islands. Cleveland.

Boxer, C. R.  (1970).  Plate es Sangre: Sidelights on the Drain of Spanish-American Silver in the Far East 1550-1700.  Philippine Studies.  18 (3), July.

Croix, Marquis de.  (1769).  Correspondence de Marquis de Croix. Croix to Marquis de Henchin. June 20.

Diaz, C.  (1890).  Conquistas de las Islas Filipinas.  Valladolid.

Gould, R. A.  (1986).  Shipwreck Anthropology.  Santa Fe.

Gould, R. A.  1986).  Looking Below the Surface: Shipwreck Archaeology as Anthropology. Santa Fe.

Hayes, J. D.  (1934).  The Manila Galleons.  US Naval Institute Proceedings. 1690-96.

Ives, R. L.  (1964).  The Manila Galleons.  The Journal of Geography.  LXIII (1).

Lagarda, B.  (1955).  Two and a Half Centuries of the Galleon Trade.  Philippine Studies.  3 (4). December.

Lenihan, D. J.  (1974).  Shipwrecks as Archaeological Phenomena:. In: Underwater Archaeology in the National Park Service. Santa Fe.

Lenihan, D. J.  (1986).  Rethinking Shipwreck Archaeology. A History of Ideas and Considerations for New Directions.  In: Gould, R. A (1986).

Le Roy,  J. A. (1905).  The Philippine Situado… American Historical Review.  10 (4). July.

Manrique, S.  (1649).  Intinerario de las Misiones que hizo al Padre…  cap 43, 285.

Morga, A. de.  (1609).  Sucesos de las islas Filipinas.  Mexico.

Murphy, L.  (1986).  Shipwrecks as Database for Human Behavioral Studies.  In: Gould (1986).

Perez, G. S.  (1954).  Manila Galleons and Mexican Pieces of Eight.  Philippines Social Sciences and Humanities Review.

Santos, J de. (1609).  Ethiopia Oriental.  parte II, livro 4, cap 2.

Schurz, W. L. (1918).  Mexico, Peru, and the Manila Galleon.  The Hispanic American Historical review. 1 (4). November.

Schurz, W. L.  (1939).  The Manila Galleon.  Cleveland, USA.

Watson, P. J.  (1986).  Method and Theory in Shipwreck Archaeology.  In: Gould (1986).

Zaide, G. F.  (1971).  Manila and Acapulco.  Philippine Historical Review.   245-70.

Postscript.

A research proposal invited for consideration and discussed at a college of the University of Oxford, April 1995. Title of investigation was ‘An Historical Maritime Anthropological Perspective of the Manila Galleon Trade.’  Research Studentship and Junior Fellowship were eventually financially unavailable.