subunit assay), pregnancy-specific a ,-glycoprotein, and cafetoprotein persist longer after some spontaneous abortions than after the birth of normal babies. Pastorfide et al"9 showed that HCG persists for about twice as long after therapeutic abortions as after normal vaginal delivery (22 days as opposed to 11) and attributed this to viable trophoblastic cells being left in the uterus after surgical intervention, since after term delivery there is a more complete removal of placental tissue.Whatever the correct explanation our findings suggest that spontaneous abortions are more relevant to ASB and other abnormalities than has been thought. Because of the short IPG in many of the index cases it might be informative to determine whether the incidence of malformations is reduced when a subsequent pregnancy is delayed after a spontaneous abortion, especially if the HCG concentration is raised. There has been a pronounced fall in the birth rate, particularly in social classes IV and V, which is probably the result of family-planning campaigns. Additional advice could easily be given about procedure after a spontaneous abortion, and if this were done and our hypothesis is correct the incidence of ASB and probably other abnormalities would automatically fall.We thank the obstetricians and paediatricians at Broadgreen, Fazakerly, the Maternity, Mill Road, and Sefton General hospitals, Liverpool, for allowing us to study their case notes, and the clerical staff in the records offices of these hospitals for much help. We are indebted to Professor Frank Harris for valuable advice, and Dr S C Rogers for useful discussions. We are grateful to the F B Baily Charitable Trust and the Nuffield Foundation for financial support.Requests for reprints should be addressed to: Sir Cyril Clarke, Neonate, 1977, 31, 84. 16 Stevenson, A C, and McClarin, R H, Nature, 1957, 180, 198. 17 Clarke, C A, et al, British Medical journal, 1976, 1, 455. 1 Roberts, C J, and Powell, R G, Lancet, 1975, 2, 848. 19 Pastorfide, Greg B, et al, American journal of Obstetrics and Gynecology, 1974, 118, 293. (Accepted 8 February 1978) Creamatocrit: simple clinical technique for estimating fat concentration and energy value of human milk A LUCAS, J A H GIBBS, R L J LYSTER, J D BAUM British Medical Journal, 1978, 1, 1018-1020 Summary and conclusions A simple micromethod has been devised for estimating the fat and energy content of human milk based on the centrifugation of milk in a haematocrit centrifuge. The percentage of cream, or "creamatocrit," is read from the haematocrit capillary tube and is linearly related to the fat and energy content.The technique, which is rapid and cheap, may be used in clinical practice, in research, and in epidemiological studies.

SUMMABY. The rates of denaturation by heat of a-lactalbumin and /Mactoglobulin in skim-milk were measured by an immunodiffusion method over a wide range of temperatures. Both reactions showed an unusual temperature dependence.The denaturation of a-lactalbumin is a first-order reaction; between 90 and 155 °C the kinetic constant k x in s -1 is given by the equationwhere T is the temperature in °K.The denaturation of /Mactoglobulin in skim-milk is second order with respect to time, and the kinetic constant k 2 in 1 g -1 s -1 is given by 2 equations, valid for different temperature ranges. Between 68 and 90 °C, log Jfc 2 = 37-95-14-51 (10 3 /T). Between 90 and 135 °C log& 2 = 5-98-2-86 (W/T).Results obtained by the immunodiffusion method agreed well with those found by salt fractionation of the milk proteins. The denaturation rates decreased when a specific reagent for sulphydryl groups was added, suggesting that such groups are involved in the denaturation of both proteins.

Quantitative polyacrylamide gel electrophoresis has been used to measure residual native whey proteins remaining after heat treatment of skim-milk and cheese whey in a kinetic study. The denaturation of a-lactalbumin (a-la) appeared to be first order, but was probably a second-order reaction displaying pseudo firstorder kinetics. The denaturation of both yff-lactoglobulin A and B (/MgA and /MgB) followed second-order kinetics while that of serum albumin was more complex, and could equally well be described as first or second order. Equations are given relating log k x (in s-1 ) to temperature for a-la denaturation in skim-milk between 70 and 95 °C and between 100 and 150 °C. Similarly, equations relating log k 2 (in lg-1^-1 ) to temperature are given for /MgA in skim-milk between 100 and 150 °C, and for yff-lgB between 95 and 150 °C. The relative heat stability of /MgA and /MgB was found to vary.Below 95 °C /MgA appeared slightly more thermostable than /MgB in skim-milk, and the same was observed in cheese whey below 100 °C. Above these temperatures yff-lgB appeared more stable than /MgA. Denaturation of /MgB was only slightly more rapid in skim-milk than in whey at temperatures below 95 °C, but was significantly slower at higher temperatures.The recovery of residual protein from cheese whey is important to the dairy industry, and the denaturation of these whey proteins is therefore of considerable interest. However, the many studies which have been reported are often not directly comparable because of the variation in experimental technique and in the means used to express results. Kinetic data on rates of denaturation are rarely available, and then are not always in agreement. Lyster (1970), using immunochemical techniques, has measured the rates of denaturation of a-lactalbumin (a-la) and /Mactoglobulin (/?-lg) in heated milk, and found a-la to follow first-order kinetics, while /Mg followed second-order kinetics. Other workers have reported the denaturation of y#-lg as first order (Gough & Jenness, 1962). A rapid accurate measure of individual native whey proteins in complex mixtures is possible using polyacrylamide gel electrophoresis (PAGE) (Hillier, 1976;Darling & Butcher, 1976). This technique has been used for a comprehensive study of the kinetics of denaturation of the major proteins in milk and cheese whey over a wide range of temperatures.

The effect of depletion of Ca2+ on the composition and size distribution of casein micelles in milk has been examined using chemical analysis, size exclusion chromatography, fast protein liquid chromatography, turbidimetry and photon correlation spectroscopy. Partial removal of Ca2 + by EDTA and subsequent dialysis resulted in disaggregation of some of the casein inicelles; as the EDTA concentration increased, the proportions of CaZ + and phosphate relative to protein in the micelles remaining intact decreased. However, the composition of the intact micelles, with respect to the different caseins, and the number-frequency size distribution were essentially unchanged.The caseins (asl, m2, / l and k--casein polypeptides) account for more than 80% of the protein in bovine milk [l]. They exist predominantly as large stable aggregates (20-600 nm diameter), which are known as casein micelles, in dynamic equilibrium with smaller aggregates and monomers [2, 31. These casein micelles contain appreciable deposits of calcium phosphate and calcium citrate (approximately 7% by mass) [2]. The calf absorbs milk calcium and phosphorus very efficiently [4] and it is likely that one important biological function of casein micelles is the transfer of calcium and phosphate to the calf in a compact but easily assimilable form. An efficient, natural, controlled release mechanism for calcium and phosphate seems to be operating in milk and it is of interest to understand the mechanism of action, which may be relevant to the devising of artificial controlled-release delivery systems for drugs and dietary supplements.While Ca2' is essential to the maintenance of casein micelle structure (the removal of Ca2+ ions from the aqueous environment results in disaggregation of the micelles [2]), it has been reported that some micellar depletion of Ca2 + is possible while retaining some structural integrity as indicated, for example, by measurement of average hydrodynamic diameter [5]. In an earlier paper [6] we described the estimation of the total refractive index of the casein micelles, which is compounded from contributions arising from the protein and inorganic constituents; this present work was begun with the intention of identifying these different contributions by selective removal of micellar calcium phosphate. However, we found that it was not possible to remove significant amounts of Ca2 + without some micellar disaggregation. The experiments described here were undertaken to examine whether, during the disaggregation, there was progressive loss of material from the micelles resulting in a decrease in micelle size.Correspondence to M. C . A. Griffin, AFRC Institute of Food Research, Reading Laboratory, Shinfield, Reading, RG2 9AT, England Abbreviations. PCS, photon correlation spectroscopy; CPG, controlled-pore glass. MATERIALS AND METHODSRaw milk was obtained from a bulk storage tank; 16 1 was centrifuged in batches at 1600 x g,, for 20 min at 5 "C, and the cream was removed. NaN3 was added to the skim milk to A method similar to tha...

SUMMARYThe irreversible heat denaturation of α-lactalbumin (α-la) in 0·1 M-phosphate, pH 7·0, at 100 °C was studied using polyacrylamide-gel electrophoresis (PAGE). PAGE revealed two groups of bands, one moving faster than native α-la and one slower, in addition to some denatured protein which remained at the origin and some residual native α-la. The faster group had unchanged molecular weight, but an increase in charge, partly due to hydrolysis of glutamine and asparagine residues. The slower group was shown by two-dimensional sodium dodecyl sulphate-PAGE to be oligomers of denatured α-la; formation of the smaller oligomers preceded the larger ones. The oligomers reverted to monomers in the presence of dithiothreitol, showing that they were disulphide-linked aggregates of denatured α-la. Immuno-blots of the gels showed that both fast and slow groups of bands had irreversibly lost most of the antigenicity of the native protein.